Metapneumovirus strains and their use in vaccine formulations and as vectors for expression of antigenic sequences

ABSTRACT

The present invention provides an isolated mammalian negative strand RNA virus,  metapneumovirus  (MPV), within the sub-family Pneumoviridae, of the family Paramyxoviridae. The invention also provides isolated mammalian negative strand RNA viruses identifiable as phylogenetically corresponding or relating to the genus  Metapneumovirus  and components thereof. In particular the invention provides a mammalian MPV, subgroups and variants thereof. The invention relates to genomic nucleotide sequences of different isolates of mammalian metapneumoviruses, in particular human metapneumoviruses. The invention relates to the use of the sequence information of different isolates of mammalian metapneumoviruses for diagnostic and therapeutic methods. The present invention relates to nucleotide sequences encoding the genome of a  metapneumovirus  or a portion thereof, including both mammalian and avian  metapneumovirus . The invention further encompasses chimeric or recombinant viruses encoded by said nucleotide sequences. The invention also relates to chimeric and recombinant mammalian MPV that comprise one or more non-native or heterologous sequences. The invention further relates to vaccine formulations comprising mammalian or avian  metapneumovirus , including recombinant and chimeric forms of said viruses. The vaccine preparations of the invention encompass multivalent vaccines, including bivalent and trivalent vaccine preparations.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/319,152, filed Dec. 31, 2008, which will issue as U.S. Pat. No.8,722,341 on May 13, 2014, which application is a continuation of U.S.patent application Ser. No. 10/371,122, filed Feb. 21, 2003, now U.S.Pat. No. 7,531,342, issued May 12, 2009, which is a continuation-in-partof International Application No: PCT/NL02/00040, filed Jan. 18, 2002,which claims priority to European Patent Application 01200213.5, filedJan. 19, 2001 and European Patent Application 01203985.5, filed Oct. 18,2001, all of which are hereby incorporated herein in their entireties bythis reference. U.S. patent application Ser. No. 10/371,122, filed Feb.21, 2003, additionally claims priority to U.S. Provisional ApplicationSer. No. 60/358,934, filed Feb. 21, 2002, the disclosure of which ishereby incorporated herein in its entirety by this reference.

Co-pending and co-assigned U.S. patent application Ser. No. 10/371,264,filed on Feb. 21, 2003, listing Ronaldus Fouchier, Bernadetta van denHoogen, Albertus Osterhaus, Aurelia Haller, and Roderick Tang asInventors, entitled “Recombinant Parainfluenza Virus Expression Systemsand Vaccines Comprising Heterologous Antigens Derived fromMetapneumovirus,” is hereby incorporated herein in its entirety by thisreference.

STATEMENT ACCORDING TO 37 C.F.R. §1.821(c) or (e) Sequence ListingSubmitted as PDF File with a Request to Transfer CRF from ParentApplication

Pursuant to 37 C.F.R. §1.821(c) or (e), a file containing a PDF versionof the Sequence Listing has been submitted concomitant with thisapplication, the contents of which are hereby incorporated by reference.The transmittal documents of this application include a Request toTransfer CRF from the parent application.

1. INTRODUCTION

The invention relates to an isolated mammalian negative strand RNAvirus, metapneumovirus (MPV), within the sub-family Pneumoviridae, ofthe family Paramyxoviridae. The present invention also relates toisolated mammalian negative strand RNA viruses identifiable asphylogenetically corresponding or relating to the genus Metapneumovirusand components thereof. The invention relates to genomic nucleotidesequences of different isolates of mammalian metapneumoviruses, inparticular human metapneumoviruses. The invention relates to the use ofthe sequence information of different isolates of mammalianmetapneumoviruses for diagnostic and therapeutic methods. The presentinvention relates to nucleotide sequences encoding the genome of ametapneumovirus or a portion thereof, including both mammalian and avianmetapneumovirus. The invention further encompasses chimeric orrecombinant viruses encoded by the nucleotide sequences. The inventionalso relates to chimeric and recombinant mammalian MPV that comprise oneor more non-native or heterologous sequences. The invention furtherrelates to vaccine formulations comprising mammalian or avianmetapneumovirus, including recombinant and chimeric forms of theviruses. The vaccine preparations of the invention encompass multivalentvaccines, including bivalent and trivalent vaccine preparations.

2. BACKGROUND OF THE INVENTION

Classically, as devastating agents of disease, paramyxoviruses accountfor many animal and human deaths worldwide each year. TheParamyxoviridae form a family within the order of Mononegavirales(negative-sense single-stranded RNA viruses), consisting of thesub-families Paramyxovirinae and Pneumovirinae. The latter sub-family isat present taxonomically divided in the genera Pneumovirus andMetapneumovirus (Pringle, 1999, Arch. Virol. 144/2, 2065-2070). Humanrespiratory syncytial virus (hRSV), a species of the Pneumovirus genus,is the single most important cause of lower respiratory tract infectionsduring infancy and early childhood worldwide (Domachowske & Rosenberg,1999, Clin. Microbio. Rev. 12(2): 298-309). Other members of thePneumovirus genus include the bovine and ovine respiratory syncytialviruses and pneumonia virus of mice (PVM).

In the past decades several etiological agents of mammalian disease, inparticular of respiratory tract illnesses (RTI), in particular ofhumans, have been identified (Evans, In: Viral Infections of Humans,Epidemiology and Control. 3th edn. (ed. Evans, A. S) 22-28 (PlenumPublishing Corporation, New York, 1989)). Classical etiological agentsof RTI with mammals are respiratory syncytial viruses belonging to thegenus Pneumovirus found with humans (hRSV) and ruminants such as cattleor sheep (bRSV and/or oRSV). In human RSV differences in reciprocalcross neutralization assays, reactivity of the G proteins inimmunological assays and nucleotide sequences of the G gene are used todefine two hRSV antigenic subgroups. Within the subgroups the amino acidsequences show 94% (subgroup A) or 98% (subgroup B) identity, while only53% amino acid sequence identity is found between the subgroups.Additional variability is observed within subgroups based on monoclonalantibodies, RT-PCR assays and RNAse protection assays. Viruses from bothsubgroups have a worldwide distribution and may occur during a singleseason. Infection may occur in the presence of pre-existing immunity andthe antigenic variation is not strictly required to allow re-infection.See, for example Sullender, 2000, Clinical Microbiology Reviews 13(1):1-15; Collins et al. Fields Virology, ed. B. N. Knipe, P. M. Howley,1996, Philadelphia: Lippencott-Raven. 1313-1351; Johnson et al., 1987(Proc. Natl. Acad. Sci. USA, 84(16): 5625-9; Collins, in TheParamyxoviruses, D. W. Kingsbury, Editor. 1991, Plenum Press: New York.p. 103-153.

Another classical Pneumovirus is the pneumonia virus of mice (PVM), ingeneral only found with laboratory mice. However, a proportion of theillnesses observed among mammals can still not be attributed to knownpathogens.

2.1 Avian Metapneumovirus

Respiratory disease caused by an avian pneumovirus (APV) was firstdescribed in South Africa in the late 1970s (Buys et al., 1980, Turkey28:36-46) where it had a devastating effect on the turkey industry. Thedisease in turkeys was characterized by sinusitis and rhinitis and wascalled turkey rhinotracheitis (TRT). The European isolates of APV havealso been strongly implicated as factors in swollen head syndrome (SHS)in chickens (O'Brien, 1985, Vet. Rec. 117:619-620). Originally, thedisease appeared in broiler chicken flocks infected with Newcastledisease virus (NDV) and was assumed to be a secondary problem associatedwith Newcastle disease (ND). Antibody against European APV was detectedin affected chickens after the onset of SHS (Cook et al., 1988, AvianPathol. 17:403-410), thus implicating APV as the cause.

Avian pneumovirus (APV) also known as turkey rhinotracheitis virus(TRTV), the etiological agent of avian rhinotracheitis, an upperrespiratory tract infection of turkeys (Giraud et al., 1986, Vet. Res.119:606-607), is the sole member of the recently assignedMetapneumovirus genus, which, as said was until now not associated withinfections, or what is more, with disease of mammals. Serologicalsubgroups of APV can be differentiated on the basis of nucleotide oramino acid sequences of the G glycoprotein and neutralization testsusing monoclonal antibodies that also recognize the G glycoprotein.However, other differences in the nucleotide and amino acid sequencescan be used to distinguish serological subgroups of APV. Withinsubgroups A, B and D, the G protein shows 98.5 to 99.7% aa sequenceidentity within subgroups while between the subgroups only 31.2-38% aaidentity is observed. See for example Collins et al., 1993, AvianPathology, 22: p. 469-479; Cook et al., 1993, Avian Pathology, 22:257-273; Bayon-Auboyer et al., J. Gen. Virol., 81(Pt 11): 2723-33; Seal,1998, Virus Res., 58(1-2): 45-52; Bayon-Auboyer et al., 1999, Arch.Virol., 144(6): 91-109; Juhasz, et al., 1994, J. Gen. Virol., 75(Pt 11):2873-80.

A further serotype of APV is provided in WO 00/20600, incorporated byreference herein, which describes the Colorado isolate of APV andcompared it to known APV or TRT strains with in vitro serumneutralization tests. First, the Colorado isolate was tested againstmonospecific polyclonal antisera to recognize TRT isolates. The Coloradoisolate was not neutralized by monospecific antisera to any of the TRTstrains. It was, however, neutralized by a hyperimmune antiserum raisedagainst a subgroup A strain. This antiserum neutralized the homologousvirus to a titer of 1:400 and the Colorado isolate to a titer of 1:80.Using the above method, the Colorado isolate was then tested against TRTmonoclonal antibodies. In each case, the reciprocal neutralization titerwas <10. Monospecific antiserum raised to the Colorado isolate was alsotested against TRT strains of both subgroups. None of the TRT strainstested were neutralized by the antiserum to the Colorado isolate.

The Colorado strain of APV does not protect SPF chicks against challengewith either a subgroup A or a subgroup B strain of TRT virus. Theseresults suggest that the Colorado isolate may be the first example of afurther serotype of avian pneumovirus (See, Bayon-Auboyer et al., 2000,J. Gen. Vir. 81:2723-2733).

The avian pneumovirus is a single-stranded, non-segmented RNA virus thatbelongs to the sub-family Pneumovirinae of the family Paramyxoviridae,genus metapneumovirus (Cavanagh and Barrett, 1988, Virus Res.11:241-256; Ling et al., 1992, J. Gen. Virol. 73:1709-1715; Yu et al.,1992, J. Gen. Virol. 73:1355-1363). The Paramyxoviridae family isdivided into two sub-families: the Paramyxovirinae and Pneumovirinae.The subfamily Paramyxovirinae includes, but is not limited to, thegenera: Paramyxovirus, Rubulavirus, and Morbillivirus. Recently, thesub-family Pneumovirinae was divided into two genera based on geneorder, and sequence homology, i.e. pneumovirus and metapneumovirus(Naylor et al., 1998, J. Gen. Virol., 79:1393-1398; Pringle, 1998, Arch.Virol. 143:1449-1159). The pneumovirus genus includes, but is notlimited to, human respiratory syncytial virus (hRSV), bovine respiratorysyncytial virus (bRSV), ovine respiratory syncytial virus, and mousepneumovirus. The metapneumovirus genus includes, but is not limited to,European avian pneumovirus (subgroups A and B), which is distinguishedfrom hRSV, the type species for the genus pneumovirus (Naylor et al.,1998, J. Gen. Virol., 79:1393-1398; Pringle, 1998, Arch. Virol.143:1449-1159). The US isolate of APV represents a third subgroup(subgroup C) within metapneumovirus genus because it has been found tobe antigenically and genetically different from European isolates (Seal,1998, Virus Res. 58:45-52; Senne et al., 1998, In: Proc. 47th WPDC,California, pp. 67-68).

Electron microscopic examination of negatively stained APV revealspleomorphic, sometimes spherical, virions ranging from 80 to 200 nm indiameter with long filaments ranging from 1000 to 2000 nm in length(Collins and Gough, 1988, J. Gen. Virol. 69:909-916). The envelope ismade of a membrane studded with spikes 13 to 15 nm in length. Thenucleocapsid is helical, 14 nm in diameter and has 7 nm pitch. Thenucleocapsid diameter is smaller than that of the genera Paramyxovirusand Morbillivirus, which usually have diameters of about 18 nm.

Avian pneumovirus infection is an emerging disease in the USA despiteits presence elsewhere in the world in poultry for many years. In May1996, a highly contagious respiratory disease of turkeys appeared inColorado, and an APV was subsequently isolated at the NationalVeterinary Services Laboratory (NVSL) in Ames, Iowa (Senne et al., 1997,Proc. 134th Ann. Mtg., AVMA, pp. 190). Prior to this time, the UnitedStates and Canada were considered free of avian pneumovirus (Pearson etal., 1993, In: Newly Emerging and Re-emerging Avian Diseases: AppliedResearch and Practical Applications for Diagnosis and Control, pp.78-83; Hecker and Myers, 1993, Vet. Rec. 132:172). Early in 1997, thepresence of APV was detected serologically in turkeys in Minnesota. Bythe time the first confirmed diagnosis was made, APV infections hadalready spread to many farms. The disease is associated with clinicalsigns in the upper respiratory tract: foamy eyes, nasal discharge andswelling of the sinuses. It is exacerbated by secondary infections.Morbidity in infected birds can be as high as 100%. The mortality canrange from 1 to 90% and is highest in six to twelve week old poults.

Avian pneumovirus is transmitted by contact. Nasal discharge, movementof affected birds, contaminated water, contaminated equipment;contaminated feed trucks and load-out activities can contribute to thetransmission of the virus. Recovered turkeys are thought to be carriers.Because the virus is shown to infect the epithelium of the oviduct oflaying turkeys and because APV has been detected in young poults, eggtransmission is considered a possibility.

2.2 PIV Infections

Parainfluenza viral infection results in serious respiratory tractdisease in infants and children. (Tao et al., 1999, Vaccine 17:1100-08). Infectious parainfluenza viral infections account forapproximately 20% of all hospitalizations of pediatric patientssuffering from respiratory tract infections worldwide. Id.

PIV is a member of the genus respirovirus (PIV1, PIV3) or rubulavirus(PIV2, PIV4) of the paramyxoviridae family. PIV is made up of twostructural modules: (1) an internal ribonucleoprotein core, ornucleocapsid, containing the viral genome, and (2) an outer, roughlyspherical lipoprotein envelope. Its genome is a single strand ofnegative sense RNA, approximately 15,456 nucleotides in length, encodingat least eight polypeptides. These proteins include, but are not limitedto, the nucleocapsid structural protein (NP, NC, or N depending on thegenera), the phosphoprotein (P), the matrix protein (M), the fusionglycoprotein (F), the hemagglutinin-neuraminidase glycoprotein (HN), thelarge polymerase protein (L), and the C and D proteins of unknownfunction. Id.

The parainfluenza nucleocapsid protein (NP, NC, or N) consists of twodomains within each protein unit including an amino-terminal domain,comprising about two-thirds of the molecule, which interacts directlywith the RNA, and a carboxyl-terminal domain, which lies on the surfaceof the assembled nucleocapsid. A hinge is thought to exist at thejunction of these two domains, thereby imparting some flexibility tothis protein (see Fields et al. (ed.), 1991, Fundamental Virology,Second Edition, Raven Press, New York, incorporated by reference hereinin its entirety). The matrix protein (M), is apparently involved withviral assembly and interacts with both the viral membrane as well as thenucleocapsid proteins. The phosphoprotein (P), which is subject tophosphorylation, is thought to play a regulatory role in transcription,and may also be involved in methylation, phosphorylation andpolyadenylation. The fusion glycoprotein (F) interacts with the viralmembrane and is first produced as an inactive precursor, then cleavedpost-translationally to produce two disulfide linked polypeptides. Theactive F protein is also involved in penetration of the parainfluenzavirion into host cells by facilitating fusion of the viral envelope withthe host cell plasma membrane. Id. The glycoprotein,hemagglutinin-neuraminidase (HN), protrudes from the envelope allowingthe virus to contain both hemagglutinin and neuraminidase activities. HNis strongly hydrophobic at its amino terminal which functions to anchorthe HN protein into the lipid bilayer. Id. Finally, the large polymeraseprotein (L) plays an important role in both transcription andreplication. Id.

2.3 RSV Infections

Respiratory syncytial virus (RSV) is the leading cause of serious lowerrespiratory tract disease in infants and children (Feigen et al., eds.,1987, In: Textbook of Pediatric Infectious Diseases, WB Saunders,Philadelphia at pages 1653-1675; New Vaccine Development, EstablishingPriorities, Vol. 1, 1985, National Academy Press, Washington D.C. atpages 397-409; and Ruuskanen et al., 1993, Curr. Probl. Pediatr.23:50-79). The yearly epidemic nature of RSV infection is evidentworldwide, but the incidence and severity of RSV disease in a givenseason vary by region (Hall, 1993, Contemp. Pediatr. 10:92-110). Intemperate regions of the northern hemisphere, it usually begins in latefall and ends in late spring. Primary RSV infection occurs most often inchildren from 6 weeks to 2 years of age and uncommonly in the first 4weeks of life during nosocomial epidemics (Hall et al., 1979, New Engl.J. Med. 300:393-396). Children at increased risk for RSV infectioninclude, but are not limited to, preterm infants (Hall et al., 1979, NewEngl. J. Med. 300:393-396) and children with bronchopulmonary dysplasia(Groothuis et al., 1988, Pediatrics 82:199-203), congenital heartdisease (MacDonald et al., New Engl. J. Med. 307:397-400), congenital oracquired immunodeficiency (Ogra et al., 1988, Pediatr. Infect. Dis. J.7:246-249; and Pohl et al., 1992, J. Infect. Dis. 165:166-169), andcystic fibrosis (Abman et al., 1988, J. Pediatr. 113:826-830). Thefatality rate in infants with heart or lung disease who are hospitalizedwith RSV infection is 3%-4% (Navas et al., 1992, J. Pediatr.121:348-354).

RSV infects adults as well as infants and children. In healthy adults,RSV causes predominantly upper respiratory tract disease. It hasrecently become evident that some adults, especially the elderly, havesymptomatic RSV infections more frequently than had been previouslyreported (Evans, A. S., eds., 1989, Viral Infections of Humans.Epidemiology and Control, 3rd ed., Plenum Medical Book, New York atpages 525-544). Several epidemics also have been reported among nursinghome patients and institutionalized young adults (A. R. Falsey, 1991,Infect. Control Hosp. Epidemiol. 12:602-608; and Garvie et al., 1980,Br. Med. J. 281:1253-1254) Finally, RSV may cause serious disease inimmunosuppressed persons, particularly bone marrow transplant patients(Hertz et al., 1989, Medicine 68:269-281).

Treatment options for established RSV disease are limited. Severe RSVdisease of the lower respiratory tract often requires considerablesupportive care, including administration of humidified oxygen andrespiratory assistance (Fields et al., eds, 1990, Fields Virology, 2nded., Vol. 1, Raven Press, New York at pages 1045-1072).

While a vaccine might prevent RSV infection, and/or RSV-related disease,no vaccine is yet licensed for this indication. A major obstacle tovaccine development is safety. A formalin-inactivated vaccine, thoughimmunogenic, unexpectedly caused a higher and more severe incidence oflower respiratory tract disease due to RSV in immunized infants than ininfants immunized with a similarly prepared trivalent parainfluenzavaccine (Kim et al., 1969, Am. J. Epidemiol. 89:422-434; and Kapikian etal., 1969, Am. J. Epidemiol. 89:405-421). Several candidate RSV vaccineshave been abandoned and others are under development (Murphy et al.,1994, Virus Res. 32:13-36), but even if safety issues are resolved,vaccine efficacy must also be improved. A number of problems remain tobe solved. Immunization would be required in the immediate neonatalperiod since the peak incidence of lower respiratory tract diseaseoccurs at 2-5 months of age. The immaturity of the neonatal immuneresponse together with high titers of maternally acquired RSV antibodymay be expected to reduce vaccine immunogenicity in the neonatal period(Murphy et al., 1988, J. Virol. 62:3907-3910; and Murphy et al., 1991,Vaccine 9:185-189). Finally, primary RSV infection and disease do notprotect well against subsequent RSV disease (Henderson et al., 1979, NewEngl. J. Med. 300:530-534).

Currently, the only approved approach to prophylaxis of RSV disease ispassive immunization. Initial evidence suggesting a protective role forIgG was obtained from observations involving maternal antibody inferrets (Prince, G. A., Ph.D. diss., University of California, LosAngeles, 1975) and humans (Lambrecht et al., 1976, J. Infect. Dis.134:211-217; and Glezen et al., 1981, J. Pediatr. 98:708-715). Hemminget al. (Morell et al., eds., 1986, Clinical Use of IntravenousImmunoglobulins, Academic Press, London at pages 285-294) recognized thepossible utility of RSV antibody in treatment or prevention of RSVinfection during studies involving the pharmacokinetics of anintravenous immune globulin (WIG) in newborns suspected of havingneonatal sepsis. In this study, it was noted that one infant, whoserespiratory secretions yielded RSV, recovered rapidly after WIGinfusion. Subsequent analysis of the WIG lot revealed an unusually hightiter of RSV neutralizing antibody. This same group of investigatorsthen examined the ability of hyperimmune serum or immune globulin,enriched for RSV neutralizing antibody, to protect cotton rats andprimates against RSV infection (Prince et al., 1985, Virus Res.3:193-206; Prince et al., 1990, J. Virol. 64:3091-3092; Hemming et al.,1985, J. Infect. Dis. 152:1083-1087; Prince et al., 1983, Infect. Immun.42:81-87; and Prince et al., 1985, J. Virol. 55:517-520). Results ofthese studies indicate that WIG may be used to prevent RSV infection, inaddition to treating or preventing RSV-related disorders.

Recent clinical studies have demonstrated the ability of this passivelyadministered RSV hyperimmune globulin (RSV WIG) to protect at-riskchildren from severe lower respiratory infection by RSV (Groothius etal., 1993, New Engl. J. Med. 329:1524-1530; and The PREVENT Study Group,1997, Pediatrics 99:93-99). While this is a major advance in preventingRSV infection, this treatment poses certain limitations in itswidespread use. First, RSV WIG must be infused intravenously overseveral hours to achieve an effective dose. Second, the concentrationsof active material in hyperimmune globulins are insufficient to treatadults at risk or most children with comprised cardiopulmonary function.Third, intravenous infusion necessitates monthly hospital visits duringthe RSV season Finally, it may prove difficult to select sufficientdonors to produce a hyperimmune globulin for RSV to meet the demand forthis product. Currently, only approximately 8% of normal donors have RSVneutralizing antibody titers high enough to qualify for the productionof hyperimmune globulin.

One way to improve the specific activity of the immunoglobulin would beto develop one or more highly potent RSV neutralizing monoclonalantibodies (MAbs). Such MAbs should be human or humanized in order toretain favorable pharmacokinetics and to avoid generating a humananti-mouse antibody response, as repeat dosing would be requiredthroughout the RSV season. Two glycoproteins, F and G, on the surface ofRSV have been shown to be targets of neutralizing antibodies (Fields etal., 1990, supra; and Murphy et al., 1994, supra).

A humanized antibody directed to an epitope in the A antigenic site ofthe F protein of RSV, SYNAGIS®, is approved for intramuscularadministration to pediatric patients for prevention of serious lowerrespiratory tract disease caused by RSV at recommended monthly doses of15 mg/kg of body weight throughout the RSV season (November throughApril in the northern hemisphere). SYNAGIS® is a composite of human(95%) and murine (5%) antibody sequences. See, Johnson et al., 1997, J.Infect. Diseases 176:1215-1224 and U.S. Pat. No. 5,824,307, the entirecontents of which are incorporated herein by reference. The human heavychain sequence was derived from the constant domains of human IgG1 andthe variable framework regions of the VH genes of Cor (Press et al.,1970, Biochem. J. 117:641-660) and Cess (Takashi et al., 1984, Proc.Natl. Acad. Sci. USA 81:194-198). The human light chain sequence wasderived from the constant domain of C_(κ) and the variable frameworkregions of the VL gene K104 with J_(κ)-4 (Bentley et al., 1980, Nature288:5194-5198). The murine sequences derived from a murine monoclonalantibody, Mab 1129 (Beeler et al., 1989, J. Virology 63:2941-2950), in aprocess which involved the grafting of the murine complementaritydetermining regions into the human antibody frameworks.

A significant portion of human respiratory disease is caused by membersof the viral sub-families Paramyxovirinae and Pneumovirinae. Theidentification of another mammalian Pneumovirinae that infects humans,hMPV, is described for the first time herein. There still remains a needfor an effective vaccine to confer protection against a variety ofviruses that result in respiratory tract infection.

Citation or discussion of a reference herein shall not be construed asan admission that such is prior art to the present invention.

3. SUMMARY OF THE INVENTION

The invention relates to an isolated mammalian negative strand RNAvirus, metapneumovirus (MPV), within the sub-family Pneumovirinae, ofthe family Paramyxoviridae. The present invention also relates toisolated mammalian negative strand RNA viruses identifiable asphylogenetically corresponding or relating to the genus Metapneumovirusand components thereof. In particular, the invention relates to amammalian MPV that is phylogenetically more closely related to a virusisolate deposited as 1-2614 with CNCM, Paris than it is related to APVtype C. In more specific embodiments, the mammalian MPV can be a variantA1, A2, B1 or B2 mammalian MPV. However, the mammalian MPVs of thepresent invention may encompass additional variants yet to beidentified, and are not limited to variants A1, A2, B1 or B2.

The invention relates to genomic nucleotide sequences of differentisolates of mammalian metapneumoviruses, in particular humanmetapneumoviruses. The invention relates to the use of the sequenceinformation of different isolates of mammalian metapneumoviruses fordiagnostic and therapeutic methods. The present invention relates to thedifferences of the genomic nucleotide sequences among the differentmetapneumovirus-isolates, and their use in the diagnostic andtherapeutic methods of the invention. In specific embodiments, thenucleotide sequence of a mammalian MPV that encodes for the N, M, F, L,P, M2-1, M2-2, SH or G ORFs may be used to identify a virus of theinvention. In other specific embodiments, the nucleotide sequence ofmammalian MPV that encodes for the N, M, F, L, P, M2-1, M2-2, SH or GORFs used to classify a mammalian MPV into variant A1, A2, B1 or B2. Ina specific embodiment, the invention relates to the use of the singlenucleotide polymorphisms (SNPs) among different metapneumovirus isolatesfor diagnostic purposes.

The invention relates to recombinant and chimeric viruses that arederived from a mammalian MPV or avian pneumovirus (APV). In accordancewith the present invention, a recombinant virus is one derived from amammalian MPV or an APV that is encoded by endogenous or native genomicsequences or non-native genomic sequences. In accordance with theinvention, a non-native sequence is one that is different from thenative or endogenous genomic sequence due to one or more mutations,including, but not limited to, point mutations, rearrangements,insertions, deletions etc., to the genomic sequence that may or may notresult in a phenotypic change. In accordance with the invention, achimeric virus of the invention is a recombinant MPV or APV whichfurther comprises a heterologous nucleotide sequence. In accordance withthe invention, a chimeric virus may be encoded by a nucleotide sequencein which heterologous nucleotide sequences have been added to the genomeor in which endogenous or native nucleotide sequences have been replacedwith heterologous nucleotide sequences. In certain embodiments, achimeric virus of the invention is derived from a MPV or APV in whichone or more of the ORFs or a portion thereof is replaced by a homologousORF or a portion thereof from another strain of metapneumovirus. In anexemplary embodiment, the ORF of the F gene of a mammalian MPV isreplaced by the ORF of the F gene of an APV. In certain otherembodiments, a chimeric virus of the invention is derived from an APV inwhich one or more of the ORFs is replaced by a homologous ORF of amammalian MPV.

The present invention relates to nucleotide sequences encoding thegenome of a metapneumovirus (including mammalian and avian strains) or aportion thereof. The present invention relates to nucleotide sequencesencoding gene products of a metapneumovirus. In particular, theinvention relates to, but is not limited to, nucleotide sequencesencoding an F protein, a G protein, an M protein, an SH protein, an Nprotein, a P protein, an M2 protein, or an L protein of a MPV. Inparticular the invention relates to nucleotide sequences encoding an Fprotein, a G protein, an M protein, an SH protein, an N protein, a Pprotein, an M2 protein, or an L protein of a variant of mammalian MPV,such as but not limited to variant A1, A2, B1 or B2 of a MPV. Thepresent invention further relates to a cDNA or RNA that encodes thegenome or a portion thereof of a metapneumovirus, including bothmammalian and avian, in addition to a nucleotide sequence which isheterologous or non-native to the viral genome. The invention furtherencompasses chimeric or recombinant viruses encoded by the cDNAs orRNAs.

The invention further relates to polypeptides and amino acid sequencesof an F protein, a G protein, an M protein, an SH protein, an N protein,a P protein, an M2 protein, or an L protein of a mammalian MPV anddifferent variants of mammalian MPV. The invention further relates toantibodies against an F protein, a G protein, an M protein, an SHprotein, an N protein, a P protein, an M2 protein, or an L protein of amammalian MPV and different variants of mammalian MPV. The antibodiescan be used for diagnostic and therapeutic methods. In certain morespecific embodiments, the antibodies are specific to mammalian MPV. Incertain embodiments, the antibodies are specific to a variant ofmammalian MPV. The invention further relates to vaccine formulations andimmunogenic compositions comprising one or more of the following: an Fprotein, a G protein, an M protein, an SH protein, an N protein, a Pprotein, an M2 protein, and/or an L protein of a mammalian MPV.

The invention further relates to vaccine formulations and immunogeniccompositions comprising mammalian or avian metapneumovirus, includingrecombinant and chimeric forms of the viruses. In particular, thepresent invention encompasses vaccine preparations comprisingrecombinant or chimeric forms of MPV and/or APV. The invention furtherrelates to vaccines comprising chimeric MPV wherein the chimeric MPVencodes one or more APV proteins and wherein the chimeric MPV optionallyadditionally expresses one or more heterologous or non-native sequences.The invention also relates to vaccines comprising chimeric APV whereinthe chimeric APV encodes one or more hMPV proteins and wherein thechimeric APV optionally additionally expresses one or more heterologousor non-native sequences. The present invention also relates tomultivalent vaccines, including bivalent and trivalent vaccines. Inparticular, multivalent vaccines of the invention encompass two or moreantigenic polypeptides expressed by the same or different pneumoviralvectors. The antigenic polypeptides of the multivalent vaccines includebut are not limited to, antigenic polypeptides of MPV, APV, PIV, RSV,influenza or another negative strand RNA virus, or another virus, suchas morbillivirus.

The invention further relates to methods for treating a respiratorytract infection in a subject. In certain embodiments, the inventionrelates to treating a respiratory tract infection in a subject byadministering to the subject a vaccine formulation comprising amammalian MPV. In specific embodiments, the methods for treating arespiratory tract infection in a subject comprise administering to thesubject a vaccine formulation or an immunogenic composition comprising arecombinant or a chimeric mammalian MPV or APV. In more specificembodiments, the recombinant or chimeric mammalian MPV is attenuated. Ina specific embodiment, the invention relates to treating a respiratorytract infection in a human patient comprising administering to the humanpatient a vaccine formulation comprising a recombinant or chimeric APV,or a nucleotide sequence encoding an F protein, a G protein, an Mprotein, an SH protein, an N protein, a P protein, an M2 protein, or anL protein of APV.

The invention provides an isolated negative-sense single-stranded RNAvirus MPV belonging to the sub-family Pneumovirinae of the familyParamyxoviridae and identifiable as phylogenetically corresponding tothe genus Metapneumovirus, wherein the virus is phylogenetically moreclosely related to a virus isolate comprising the nucleotide sequence ofSEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:21 than it isrelated to turkey rhinotracheitis virus, the etiological agent of avianrhinotracheitis. In certain embodiments, the invention provides anisolated negative-sense single-stranded RNA metapneumovirus, wherein thegenome of the virus comprises a nucleotide sequence of SEQ ID NO:18. Incertain embodiments, the invention provides an isolated negative-sensesingle-stranded RNA metapneumovirus, wherein the genome of the viruscomprises a nucleotide sequence of SEQ ID NO:19. In certain embodiments,the invention provides an isolated negative-sense single-stranded RNAmetapneumovirus, wherein the genome of the virus comprises a nucleotidesequence of SEQ ID NO:20. In certain embodiments, the invention providesan isolated negative-sense single-stranded RNA metapneumovirus, whereinthe genome of the virus comprises a nucleotide sequence of SEQ ID NO:21.In certain embodiments, the invention provides an isolated nucleic acid,wherein the nucleic acid has a nucleotide sequence that is at least 70%identical to SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20 or SEQ ID NO:21,wherein sequence identity is determined over the entire length of SEQ IDNO:19, SEQ ID NO:20, SEQ ID NO:21 or SEQ ID NO:22. In certainembodiments, the invention provides an isolated nucleic acid, whereinthe nucleic acid encodes a protein comprising (i) an amino acid sequencethat is at least 66% identical to the G protein of a mammalian MPVvariant B1 (SEQ ID NO:324); (ii) an amino acid sequence that is at least98.5% identical to the N protein of a mammalian MPV variant B1 (SEQ IDNO:368); (iii) an amino acid sequence that is at least 96% identical theP protein of a mammalian MPV variant 131 (SEQ ID NO:376); (iv) an aminoacid sequence that is identical the M protein of a mammalian MPV variantB1 (SEQ ID NO:360); (v) an amino acid sequence that is at least 99%identical the F protein of a mammalian MPV variant B1 (SEQ ID NO:316);(vi) an amino acid sequence that is at least 98% identical the M2-1protein of a mammalian MPV variant B1 (SEQ ID NO:340); (vii) an aminoacid sequence that is at least 99% identical the M2-2 protein of amammalian MPV variant B1 (SEQ ID NO:348); (viii) an amino acid sequencethat is at least 83% identical the SH protein of a mammalian MPV variantB1 (SEQ ID NO:384); or (ix) an amino acid sequence that is at least 99%identical the L protein a mammalian MPV variant B1 (SEQ ID NO:332). Incertain embodiments, the invention provides an isolated nucleic acid,wherein the nucleic acid encodes a protein comprising (i) an amino acidsequence that is at least 66% identical to the G protein of a mammalianMPV variant A1 (SEQ ID NO:322); (ii) an amino acid sequence that is atleast 99.5% identical to the N protein of a mammalian MPV variant A1(SEQ ID NO:366); (iii) an amino acid sequence that is at least 96%identical to the P protein of a mammalian MPV variant A1 (SEQ IDNO:374); (iv) an amino acid sequence that is at least 99% identical tothe M protein of a mammalian MPV variant A1 (SEQ ID NO:358); (v) anamino acid sequence that is at least 98% identical to the F protein of amammalian MPV variant A1 (SEQ ID NO:314); (vi) an amino acid sequencethat is at least 99% identical to the M2-1 protein of a mammalian MPVvariant A1 (SEQ ID NO:338) (vii) an amino acid sequence that is at least96% identical to the M2-2 protein of a mammalian MPV variant A1 (SEQ IDNO:346) (viii) an amino acid sequence that is at least 84% identical tothe SH protein of a mammalian MPV variant A1 (SEQ ID NO:382); or (ix) anamino acid sequence that is at least 99% identical to the L protein of avirus of a mammalian MPV variant A1 (SEQ ID NO:330). In certainembodiments, the invention provides an isolated nucleic acid, whereinthe nucleic acid encodes a protein comprising (i) an amino acid sequencethat is at least 66% identical to the G protein of a mammalian MPVvariant A2 (SEQ ID NO:332); (ii) an amino acid sequence that is at least99.5% identical to the N protein of a mammalian MPV variant A2 (SEQ IDNO:367); (iii) an amino acid sequence that is at least 96% identical tothe P protein of a mammalian MPV variant A2 (SEQ ID NO:375); (iv) anamino acid sequence that is at least 99% identical to the M protein of amammalian MPV variant A2 (SEQ ID NO:359); (v) an amino acid sequencethat is at least 98% identical to the F protein of a mammalian MPVvariant A2 (SEQ ID NO:315); (vi) an amino acid sequence that is at least99% identical to the M2-1 protein of a mammalian MPV variant A2 (SEQ IDNO:339); (vii) an amino acid sequence that is at least 96% identical tothe M2-2 protein of a mammalian MPV variant A2 (SEQ ID NO:347); (viii)an amino acid sequence that is at least 84% identical to the SH proteinof a mammalian MPV variant A2 (SEQ ID NO:383); or (ix) an amino acidsequence that is at least 99% identical to the L protein of a mammalianMPV variant A2 (SEQ ID NO:331). In certain embodiments, the inventionprovides an isolated nucleic acid, wherein the nucleic acid encodes aprotein comprising (i) an amino acid sequence that is at least 66%identical to the G protein of a mammalian MPV variant B2 (SEQ IDNO:325); (ii) an amino acid sequence that is at least 97% identical tothe N protein of a mammalian MPV variant B2 (SEQ ID NO:369); (iii) anamino acid sequence that is at least 96% identical to the P protein of amammalian MPV variant B2 (SEQ ID NO:377); (iv) an amino acid sequencethat is identical to the M protein of a mammalian MPV variant B2 (SEQ IDNO:361) (v) an amino acid sequence that is at least 99% identical to theF protein of a mammalian MPV variant B2 (SEQ ID NO:317); (vi) an aminoacid sequence that is at least 98% identical to the M2-1 protein of amammalian MPV variant B2 (SEQ ID NO:341); (vii) an amino acid sequencethat is at least 99% identical to the M2-2 protein of a mammalian MPVvariant B2 (SEQ ID NO:349); (viii) an amino acid sequence that is atleast 84% identical to the SH protein of a mammalian MPV variant B2 (SEQID NO:385); or (ix) an amino acid sequence that is at least 99%identical to the L protein of a mammalian MPV variant B2 (SEQ IDNO:333). In certain embodiments, the invention provides an isolatednucleic acid, wherein the nucleic acid hybridizes specifically underhigh stringency, medium stringency, or low stringency conditions to anucleic acid of a mammalian MPV.

In certain embodiments, the invention provides a virus comprising thenucleotide sequence of SEQ ID NOS:18-21 or a fragment thereof.

In certain embodiments, the invention provides an isolated protein,wherein the protein comprises (i) an amino acid sequence that is atleast 66% identical to the G protein of a mammalian MPV variant B1 (SEQID NO:324); (ii) an amino acid sequence that is at least 98.5% identicalto the N protein of a mammalian MPV variant B1 (SEQ ID NO:368); (iii) anamino acid sequence that is at least 96% identical the P protein of amammalian MPV variant B1 (SEQ ID NO:376); (iv) an amino acid sequencethat is identical the M protein of a mammalian MPV variant B1 (SEQ IDNO:360); (v) an amino acid sequence that is at least 99% identical the Fprotein of a mammalian MPV variant B1 (SEQ ID NO:316) (vi) an amino acidsequence that is at least 98% identical the M2-1 protein of a mammalianMPV variant B1 (SEQ ID NO:340); (vii) an amino acid sequence that is atleast 99% identical the M2-2 protein of a mammalian MPV variant B1 (SEQID NO:348); (viii) an amino acid sequence that is at least 83% identicalthe SH protein of a mammalian MPV variant B1 (SEQ ID NO:384); or (ix) anamino acid sequence that is at least 99% identical the L protein amammalian MPV variant B1 (SEQ ID NO:332). In certain embodiments, theinvention provides an isolated protein, wherein the protein comprises:(i) an amino acid sequence that is at least 66% identical to the Gprotein of a mammalian MPV variant A1 (SEQ ID NO:322); (ii) an aminoacid sequence that is at least 99.5% identical to the N protein of amammalian MPV variant A1 (SEQ ID NO:366) (iii) an amino acid sequencethat is at least 96% identical to the P protein of a mammalian MPVvariant A1 (SEQ ID NO:374); (iv) an amino acid sequence that is at least99% identical to the M protein of a mammalian MPV variant A1 (SEQ IDNO:358); (v) an amino acid sequence that is at least 98% identical tothe F protein of a mammalian MPV variant A1 (SEQ ID NO:314); (vi) anamino acid sequence that is at least 99% identical to the M2-1 proteinof a mammalian MPV variant A1 (SEQ ID NO:338) (vii) an amino acidsequence that is at least 96% identical to the M2-2 protein of amammalian MPV variant A1 (SEQ ID NO:346) (viii) an amino acid sequencethat is at least 84% identical to the SH protein of a mammalian MPVvariant A1 (SEQ ID NO:382); or (ix) an amino acid sequence that is atleast 99% identical to the L protein of a virus of a mammalian MPVvariant A1 (SEQ ID NO:330) In certain embodiments, the inventionprovides isolated protein, wherein the protein comprises (i) an aminoacid sequence that is at least 66% identical to the G protein of amammalian MPV variant A2 (SEQ ID NO:332); (ii) an amino acid sequencethat is at least 99.5% identical to the N protein of a mammalian MPVvariant A2 (SEQ ID NO:367); (iii) an amino acid sequence that is atleast 96% identical to the P protein of a mammalian MPV variant A2 (SEQID NO:375) (iv) an amino acid sequence that is at least 99% identical tothe M protein of a mammalian MPV variant A2 (SEQ ID NO:359); (v) anamino acid sequence that is at least 98% identical to the F protein of amammalian MPV variant A2 (SEQ ID NO:315) (vi) an amino acid sequencethat is at least 99% identical to the M2-1 protein of a mammalian MPVvariant A2 (SEQ ID NO:339); (vii) an amino acid sequence that is atleast 96% identical to the M2-2 protein of a mammalian MPV variant A2(SEQ ID NO:347) (viii) an amino acid sequence that is at least 84%identical to the SH protein of a mammalian MPV variant A2 (SEQ IDNO:383); or (ix) an amino acid sequence that is at least 99% identicalto the L protein of a mammalian MPV variant A2 (SEQ ID NO:331). Incertain embodiments, the invention provides an isolated protein, whereinthe protein comprises: (i) an amino acid sequence that is at least 66%identical to the G protein of a mammalian MPV variant B2 (SEQ IDNO:325); (ii) an amino acid sequence that is at least 97% identical tothe N protein of a mammalian MPV variant B2 (SEQ ID NO:369) (iii) anamino acid sequence that is at least 96% identical to the protein of amammalian MPV variant B2 (SEQ ID NO:377) (iv) an amino acid sequencethat is identical to the M protein of a mammalian MPV variant B2 (SEQ IDNO:361); (v) an amino acid sequence that is at least 99% identical tothe F protein of a mammalian MPV variant B2 (SEQ ID NO:317); (vi) anamino acid sequence that is at least 98% identical to the M2-1 proteinof a mammalian MPV variant B2 (SEQ ID NO:341); (vii) an amino acidsequence that is at least 99% identical to the M2-2 protein of amammalian MPV variant B2 (SEQ ID NO:349); (viii) an amino acid sequencethat is at least 84% identical to the SH protein of a mammalian MPVvariant B2 (SEQ ID NO:385); or (ix) an amino acid sequence that is atleast 99% identical to the L protein of a mammalian MPV variant 132 (SEQID NO:333). In certain embodiments, the invention provides an antibody,wherein the antibody binds specifically to a protein consisting of (i)an amino acid sequence that is at least 66% identical to the G proteinof a mammalian MPV variant B1 (SEQ ID NO:324); (ii) an amino acidsequence that is at least 98.5% identical to the N protein of amammalian MPV variant B1 (SEQ ID NO:368); (iii) an amino acid sequencethat is at least 96% identical the P protein of a mammalian MPV variantB1 (SEQ ID NO:376) (iv an amino acid sequence that is identical the Mprotein of a mammalian MPV variant B1 (SEQ ID NO:360); (v) an amino acidsequence that is at least 99% identical the F protein of a mammalian MPVvariant B1 (SEQ ID NO:316); (vi) an amino acid sequence that is at least98% identical the M2-1 protein of a mammalian MPV variant B1 (SEQ IDNO:340) (vii) an amino acid sequence that is at least 99% identical theM2-2 protein of a mammalian MPV variant B1 (SEQ ID NO:348); (viii) anamino acid sequence that is at least 83% identical the SH protein of amammalian MPV variant B1 (SEQ ID NO:384); (ix) an amino acid sequencethat is at least 99% identical the L protein a mammalian MPV variant B1(SEQ ID NO:332). In certain embodiments, the invention provides anantibody, wherein the antibody binds specifically to a proteinconsisting of: (i) an amino acid sequence that is at least 66% identicalto the G protein of a mammalian MPV variant A1 (SEQ ID NO:322); (ii) anamino acid sequence that is at least 99.5% identical to the N protein ofa mammalian MPV variant A1 (SEQ ID NO:366); (iii an amino acid sequencethat is at least 96% identical to the P protein of a mammalian MPVvariant A1 (SEQ ID NO:374); (iv) an amino acid sequence that is at least99% identical to the M protein of a mammalian MPV variant A1 (SEQ IDNO:358); (v) an amino acid sequence that is at least 98% identical tothe F protein of a mammalian MPV variant A1 (SEQ ID NO:314); (vi) anamino acid sequence that is at least 99% identical to the M2-1 proteinof a mammalian MPV variant A1 (SEQ ID NO:338); (vii) an amino acidsequence that is at least 96% identical to the M2-2 protein of amammalian MPV variant A1 (SEQ ID NO:346); (viii) an amino acid sequencethat is at least 84% identical to the SH protein of a mammalian MPVvariant A1 (SEQ ID NO:382); (ix) an amino acid sequence that is at least99% identical to the L protein of a virus of a mammalian MPV variant A1(SEQ ID NO:330). In certain embodiments, the invention provides anantibody, wherein the antibody binds specifically to a proteinconsisting of (i) an amino acid sequence that is at least 66% identicalto the G protein of a mammalian MPV variant A2 (SEQ ID NO:332); (ii) anamino acid sequence that is at least 96% identical to the N protein of amammalian MPV variant A2 (SEQ ID NO:367) (iii) an amino acid sequencethat is at least 96% identical to the protein of a mammalian MPV variantA2 (SEQ ID NO:375); (iv) an amino acid sequence that is at least 99%identical to the M protein of a mammalian MPV variant A2 (SEQ IDNO:359); (v) an amino acid sequence that is at least 98% identical tothe F protein of a mammalian MPV variant A2 (SEQ ID NO:315) (vi) anamino acid sequence that is at least 99% identical to the M2-1 proteinof a mammalian MPV variant A2 (SEQ ID NO:339); (vii) an amino acidsequence that is at least 96% identical to the M2-2 protein of amammalian MPV variant A2 (SEQ ID NO:347); (viii) an amino acid sequencethat is at least 84% identical to the SH protein of a mammalian MPVvariant A2 (SEQ ID NO:383); (ix) an amino acid sequence that is at least99% identical to the L protein of a mammalian MPV variant A2 (SEQ IDNO:331) In certain embodiments, the invention provides an antibody,wherein the antibody binds specifically to a protein consisting of: (i)an amino acid sequence that is at least 66% identical to the G proteinof a mammalian MPV variant B2 (SEQ ID NO:325); (ii) an amino acidsequence that is at least 97% identical to the N protein of a mammalianMPV variant B2 (SEQ ID NO:369); (iii) an amino acid sequence that is atleast 96% identical to the P protein of a mammalian MPV variant B2 (SEQID NO:377) (iv) an amino acid sequence that is identical to the Mprotein of a mammalian MPV variant B2 (SEQ ID NO:361); (v) an amino acidsequence that is at least 99% identical to the F protein of a mammalianMPV variant B2 (SEQ ID NO:317); (vi) an amino acid sequence that is atleast 98% identical to the M2-1 protein of a mammalian MPV variant B2(SEQ ID NO:341); (vii) an amino acid sequence that is at least 99%identical to the M2-2 protein of a mammalian MPV variant B2 (SEQ IDNO:349) (viii) an amino acid sequence that is at least 84% identical tothe SH protein of a mammalian MPV variant B2 (SEQ ID NO:385); or (ix) anamino acid sequence that is at least 99% identical to the L protein of amammalian MPV variant B2 (SEQ ID NO:333). In certain embodiments, theinvention provides a method for detecting a variant B1 mammalian MPV ina sample, wherein the method comprises contacting the sample with theantibody of specific to a variant B1. In certain embodiments, theinvention provides method for detecting a variant A1 mammalian MPV in asample, wherein the method comprises contacting the sample with theantibody specific to variant A1. In certain embodiments, the inventionprovides a method for detecting a variant A2 mammalian MPV in a sample,wherein the method comprises contacting the sample with the antibodyspecific to variant A2. In certain embodiments, the invention provides amethod for detecting a variant B2 mammalian MPV in a sample, wherein themethod comprises contacting the sample with the antibody specific to B2.

In certain embodiments, the invention provides a method for identifyinga viral isolate as a mammalian MPV, wherein the method comprisescontacting the isolate or a component thereof with the antibody specificto a mammalian MPV. In certain embodiments, the invention providesmethod for virologically diagnosing a MPV infection of a mammalcomprising determining in a sample of the mammal the presence of a viralisolate or component thereof by contacting the sample with the antibodyspecific to a MPV. In certain embodiments, the invention provides methodfor virologically diagnosing a mammalian MPV infection of a subject,wherein the method comprises obtaining a sample from the subject andcontacting the sample with an antibody specific to MPV wherein if theantibody binds to the sample the subject is infected with mammalian MPV.

In certain embodiments, the invention provides an infectious recombinantvirus, wherein the recombinant virus comprises the genome of a mammalianMPV and further comprises a non-native MPV sequence. In certainembodiments, the invention provides a recombinant nucleic acid, whereinthe recombinant nucleic acid comprises (i) a nucleic acid encoding a Gpolypeptide of an MPV A1 variant; and (ii) a nucleic acid encoding anon-native MPV polypeptide. In certain embodiments, the inventionprovides recombinant nucleic acid, wherein the recombinant nucleic acidcomprises (i) a nucleic acid encoding a G polypeptide of an MPV A2variant; and (ii) a nucleic acid encoding a non-native MPV polypeptide.In certain embodiments, the invention provides s recombinant nucleicacid, wherein the recombinant nucleic acid comprises (i) a nucleic acidencoding a G polypeptide of an MPV B1 variant; and (ii) a nucleic acidencoding a non-native MPV polypeptide. In certain embodiments, theinvention provides a recombinant nucleic acid, wherein the recombinantnucleic acid comprises (i) a nucleic acid encoding a G polypeptide of anMPV B2 variant; and (ii) a nucleic acid encoding a non-native MPVpolypeptide.

In certain embodiments, the invention provides an infectious chimericvirus, wherein the chimeric virus comprises the genome of a mammalianMPV of a first variant, wherein one or more of the open reading framesin the genome of the mammalian MPV of the first variant have beenreplaced by the analogous open reading frame from a mammalian MPV of asecond variant. In certain embodiments, the invention provides aninfectious chimeric virus, wherein the chimeric virus comprises thegenome of a mammalian MPV of a first variant, wherein one or more ofopen reading frames of a mammalian MPV of a second variant are insertedinto the genome of the mammalian MPV of the first variant.

In certain embodiments, the invention provides an infectious chimericvirus, wherein the chimeric virus comprises the genome of a mammalianMPV, wherein one or more of the open reading frames in the genome of themammalian MPV have been replaced by an ORF which encodes one or more ofan avian MPV F protein; an avian MPV G protein (iii) an avian MPV SHprotein; (iv) an avian MPV N protein (v) an avian MPV P protein; (vi) anavian MPV M2 protein; (vii) an avian MPV M2-1 protein; (viii) an avianMPV M2-2 protein; or (ix) an avian MPV L protein. In certainembodiments, the invention provides an infectious chimeric virus,wherein the chimeric virus comprises the genome of an avian MPV, whereinone or more of the open reading frames in the genome of the avian MPVhave been replaced by an ORF which encodes one or more of (i) amammalian MPV F protein (ii) a mammalian MPV G protein; (iii) amammalian MPV SH protein; (iv) a mammalian MPV N protein; (v) amammalian MPV P protein; (vi) a mammalian MPV M2 protein; (vii) amammalian MPV M2-1 protein; (viii) a mammalian MPV M2-2 protein; or (ix)a mammalian MPV L protein.

In certain embodiments, the invention provides an immunogeniccomposition, wherein the immunogenic composition comprises theinfectious recombinant virus of the invention.

In certain embodiments, the invention provides a method for detecting amammalian MPV in a sample, wherein the method comprises contacting thesample with a nucleic acid sequence of the invention. In certainembodiments, the invention provides a pharmaceutical composition,wherein the pharmaceutical composition comprises the infectiousrecombinant virus of the invention.

In certain embodiments, the invention provides a method for treating orpreventing a respiratory tract infection in a mammal, the methodcomprising administering a vaccine comprising a mammalianmetapneumovirus.

In certain embodiments, the invention provides a method for treating orpreventing a respiratory tract infection in a mammal, the methodcomprising administering a vaccine comprising the recombinant mammalianmetapneumovirus of the invention.

In certain embodiments, the invention provides a method for treating orpreventing a respiratory tract infection in a mammal, the methodcomprising administering a vaccine comprising avian metapneumovirus. Incertain embodiments, the invention provides a method for treating orpreventing a respiratory tract infection in a human, the methodcomprising administering a vaccine comprising avian metapneumovirus. Incertain embodiments, the invention provides a method for treating orpreventing a respiratory tract infection in a subject, the methodcomprising administering to the subject the composition of theinvention.

In certain embodiments, the invention provides a method for identifyinga compound useful for the treatment of infections with mammalian MPV,wherein the method comprises: (a) infecting an animal with a mammalianMPV; (b) Administering to the animal a test compound; and (c)determining the effect of the test compound on the infection of theanimal, wherein a test compound that reduces the extent of the infectionor that ameliorates the symptoms associated with the infection isidentified as a compound useful for the treatment of infections withmammalian MPV. In certain embodiments, the invention provides a methodfor identifying a compound useful for the treatment of infections withmammalian MPV, wherein the method comprises (a) infecting a cell culturewith a mammalian MPV (b) incubating the cell culture with a testcompound; and (c) determining the effect of the test compound on theinfection of the cell culture, wherein a test compound that reduces theextent of the infection is identified as a compound useful for thetreatment of infections with mammalian MPV. In certain embodiments, theinvention provides a method for diagnosing a mammalian MPV infection ofan animal, wherein the method comprises determining in a sample of theanimal the presence of a viral isolate or component thereof by reactingthe sample with a nucleic acid or an antibody reactive with a componentof an avian pneumovirus, the nucleic acid or antibody beingcross-reactive with a component of MPV.

In certain embodiments, the invention provides a method forserologically diagnosing a mammalian MPV infection of an animal, whereinthe method comprises contacting a sample from the animal with theprotein of the invention. In certain embodiments, the invention providesa method for serologically diagnosing a mammalian MPV infection of ananimal, wherein the method comprises contacting a sample from the animalwith a protein of an APV. In certain embodiments, the invention providesa method for diagnosing an APV infection of a bird comprising contactinga sample from the animal with the protein of the invention.

In certain embodiments, the invention provides an isolatednegative-sense single-stranded RNA virus MPV belonging to the sub-familyPneumovirinae of the family Paramyxoviridae and identifiable asphylogenetically corresponding to the genus Metapneumovirus, wherein thevirus is phylogenetically more closely related to a virus isolatedeposited as 1-2614 with CNCM, Paris than to turkey rhinotracheitisvirus, the etiological agent of avian rhinotracheitis.

3.1 Conventions and Abbreviations

-   -   cDNA complementary DNA    -   L large protein    -   M matrix protein (lines inside of envelope)    -   F fusion glycoprotein    -   HN hemagglutinin-neuraminidase glycoprotein    -   N, NP or NC nucleoprotein (associated with RNA and required for        polymerase activity)    -   P phosphoprotein    -   MOI multiplicity of infection    -   NA neuraminidase (envelope glycoprotein)    -   PIV parainfluenza virus    -   hPIV human parainfluenza virus    -   hPIV3 human parainfluenza virus type 3    -   APV/hMPV recombinant APV with hMPV sequences    -   hMPV/APV recombinant hMPV with APV sequences    -   Mammalian MPV mammalian metapneumovirus    -   nt nucleotide    -   RNP ribonucleoprotein    -   rRNP recombinant RNP    -   vRNA genomic virus RNA    -   cRNA antigenomic virus RNA    -   hMPV human metapneumovirus    -   APV avian pneumovirus    -   MVA modified vaccinia virus Ankara    -   FACS Fluorescence Activated Cell Sorter    -   CPE cytopathic effects    -   Position 1 Position of the first gene of the viral genome to be        transcribed    -   Position 2 Position between the first and the second open        reading frame of the native viral genome, or alternatively, the        position of the second gene of the viral genome to be        transcribed    -   Position 3 Position between the second and the third open        reading frame of the native viral genome, or alternatively, the        position of the third gene of the viral genome to be        transcribed.    -   Position 4 Position between the third and the fourth open        reading frame of the native viral genome, or alternatively, the        position of the fourth gene of the viral genome to be        transcribed.    -   Position 5 Position between the fourth and the fifth open        reading frame of the native viral genome, or alternatively, the        position of the fifth gene of the viral genome to be        transcribed.    -   Position 6 Position between the fifth and the sixth open reading        frame of the native viral genome, or alternatively, the position        of the sixth gene of the viral genome to be transcribed.

3.2 Deposit of Biological Material

Mammalian metapneumovirus isolate NL/1/00 “MPV-isolate 00-1” has beendeposited with the international depository authority CollectionNationale de Cultures de Microorganismes (CNCM) as deposit accessionnumber 1-2614. The address of the CNCM is Institut Pasteur, 26, Rue duDocteur Roux, F-75724 Paris Cedex 15, France. The deposits were receivedon Jan. 19, 2001.

4. DESCRIPTION OF THE FIGURES

FIG. 1: Percentage homology found between the amino acid sequence ofisolate 00-1 and other members of the Pneumovirinae. Percentages (×100)are given for the amino acid sequences of N, P, M, F and two RAP-PCRfragments in L (8 and 9/10).

FIG. 2: Seroprevalence of MPV in humans categorized by age group, usingimmunofluorescence and virus neutralization assays.

FIG. 3: Schematic representation of the genome of APV with the locationand size of the fragments obtained with RAP-PCR and RT-PCR on virusisolate 00-1 (A1). Fragments 1 to 10 were obtained using RAP-PCR.Fragment A was obtained with a primer in RAP-PCR fragment 1 and 2 and aprimer that was designed based on alignment of leader and trailersequences of APV and RSV (Randhawa et al., 1997, J. Virol.71:9849-9854). Fragment B was obtained using primers designed in RAP-PCRfragment 1 and 2 and RAP-PCR fragment 3. Fragment C was obtained withprimers designed in RAP-PCR fragment 3 and RAP-PCR fragments 4, 5, 6,and 7.

FIGS. 4A-4E: Comparison of the N (SEQ ID NOS:390-396), P (SEQ IDNOS:397-402), M (SEQ ID NOS:403-409) and F (SEQ ID NOS:410-416) ORFs ofmembers of the subfamily Pneumovirinae and virus isolate 00-1 (A1). Thealignment shows the amino acid sequence of the complete N, F, M and Pproteins and partial L proteins of virus isolate 00-1 (A1) Amino acidsthat differ between isolate 00-1 (A1) and the other viruses are shown,identical amino acids are represented by periods. Gaps are representedas dashes. Numbers correspond to amino acid positions in the proteins.Abbreviations are as follows: APV-A, B or C: Avian Pneumovirus type A, Bor C; hRSV: bovine or human respiratory syncytial virus; PVM: pneumoniavirus of mice. L8: fragment 8 obtained with RAP-PCR located in L, L9/10: consensus of fragment 9 and 10 obtained with RAP-PCR, located in L(SEQ ID NO:417). For the L alignment only bRSV, hRSV and APV-A sequenceswere available (SEQ ID NOS:418-420).

FIG. 5: Alignment of the predicted amino acid sequence of thenucleoprotein of MPV with those of other pneumoviruses (SEQ IDNOS:421-428). The conserved regions are represented by boxes and labeledA, B, and C. The conserved region among pneumoviruses is shown in grayand shaded. Gaps are represented by dashes, periods indicate thepositions of identical amino acid residues compared to MPV.

FIG. 6: Amino acid sequence comparison of the phosphoprotein of MPV withthose of other pneumoviruses (SEQ ID NOS:429-436). The region of highsimilarity is boxed, and the glutamate rich region is in grey andshaded. Gaps are represented by dashes. Periods indicate the position ofidentical amino acid residues compared to MPV.

FIG. 7: Comparison of the deduced amino acid sequence of the matrixprotein of MPV with those of other pneumoviruses (SEQ ID NOS:437-444).The conserved hexapeptide sequence is in grey and shaded. Gaps arerepresented by dashes. Periods indicate the position of identical aminoacid residues relative to MPV.

FIG. 8: Genomic map of MPV isolate 00-1 (A1). The nucleotide positionsof the start and stop codons are indicated under each ORF. The doublelines which cross the L ORF indicate the shortened representation of theL gene. The three reading frames below the map indicate the primary GORF (nt 6262-6972) and overlapping potential secondary ORFs.

FIG. 9: Alignment of the predicted amino acid sequence of the fusionprotein of MPV with those of other pneumoviruses (SEQ ID NOS:445-452).The conserved cysteine residues are boxed. N-linked glycosylation sitesare underlined. The cleavage site of F0 is double underlined; the fusionpeptide, signal peptide, and membrane anchor domain are shown in greyand shaded. Gaps are represented by dashes, and periods indicate theposition of identical amino acids relative to MPV.

FIG. 10: Comparison of amino acid sequences of the M2 ORFs of MPV withthose of other pneumoviruses. The alignment of M2-1 ORFs is shown inpanel A (SEQ ID NOS:453-460), with the conserved amino terminus shown ingrey and shaded. The three conserved cysteine residues are printed boldface and indicated by #. The alignment of the M2-2 ORFs is shown inpanel B (SEQ ID NOS:461-468). Gaps are represented by dashes and periodsindicate the position of identical amino acids relative to MPV.

FIG. 11: Amino acid sequence analyses of the SH ORF of MPV. (A) Aminoacid sequence of the SH ORF of MPV (SEQ ID NO:469), with the serine andthreonine residues in grey and shaded, cysteine residues in bold face,and the hydrophobic region doubly underlined. Potential N-linkedglycosylation sites are single underlined. Arrows indicate the positionsof the basic amino acids flanking the hydrophobic domain. (B) Alignmentof the hydrophobicity plots of the SH proteins of MPV, APV-A and hRSV-B.A window of 17 amino acids was used. Arrows indicate a stronghydrophobic domain. Positions within the ORF are given on the X-axis.

FIG. 12: Amino acid sequence analyses of the G ORF of MPV. (A) Aminoacid sequence of the G ORF of MTV (SEQ ID NO:470), with serine,threonine, and proline residues in grey and shaded. The cysteine residueis in bold face, and the hydrophobic region is doubly underlined. Thepotential N-linked glycosylation sites are singly underlined. (B)Alignment of the hydrophobicity plots of the G proteins of MPV, APV-Aand hRSV-B. A window of 17 amino acids was used. Arrows indicate thehydrophobic region, and positions within the ORF are given at theX-axis.

FIG. 13: Comparison of the amino acid sequences of a conserved domain ofthe polymerase gene of MPV and other paramyxoviruses (SEQ IDNOS:471-481). Domain III is shown with the four conserved polymerasemotifs (A, B, C, D) in domain III (Poch et al., 1989 EMBO J. 8:3867-74;Poch et al., 1990, J. Gen. Virol 71:1153-62) boxed. Gaps are representedby dashes and periods indicate the position of identical amino acidresidues relative to MPV. Abbreviations used are as follows: hPIV-3:human parainfluenza virus type 3; SV: sendai virus; hPIV-2: humanparainfluenza virus type 2; NDV: New castle disease virus; MV: measlesvirus; nipah: Nipah virus.

FIG. 14: Phylogenetic analyses of the N, F, M, and F ORF s of members ofthe genus Pneumovirinae and virus isolate 00-1 (A1). Phylogeneticanalysis was performed on viral sequences from the following genes: F(panel A), N (panel B), M (panel C), and P (panel D). The phylogenetictrees are based on maximum likelihood analyses using 100 bootstraps and3 jumbles. The scale representing the number of nucleotide changes isshown for each tree.

FIG. 15: Phylogenetic analyses of the M2-1 and L ORFs of MPV andselected paramyxoviruses. The M2-1ORF was aligned with the M2-1 ORFs ofother members of the genus Pneumovirinae (see Panel A) and the L ORF wasaligned with L ORFs members of the genus Pneumovirinae and selectedother paramyxoviruses as described in the legend of FIG. 13.Phylogenetic trees were generated by maximum likelihood analyses using100 bootstraps and 3 jumbles. The scale representing the number ofnucleotide changes is shown for each tree. Numbers in the treesrepresent bootstrap values based on the consensus trees.

FIG. 16: Phylogenetic relationship for parts of the F (panel A), N(panel B), M (panel C) 20 and L (panel D) ORFs of nine of the primaryMPV isolates with APV-C, its closest relative genetically. Thephylogenetic trees are based on maximum likelihood analyses. The scalerepresenting the number of nucleotide changes is shown for each tree.Accession numbers for APV-C: panel A: D00850; panel B: U39295; panel C:X58639; and panel D: U65312.

FIGS. 17A-17K: Alignment of the F genes of different isolates of hMPV ofall four variants, variant A1, A2, B1, or B2 (SEQ ID NOS:154-233).

FIGS. 18A-18D: Alignment of the F proteins of different isolates of hMPVof all four variants, variant A1, A2, B1, or B2 (SEQ ID NOS:234-313).

FIGS. 19A-19J: Alignment of the G genes of different isolates of hMPV ofall four variants, variant A1, A2, B1, or B2 (SEQ ID NOS:85-118).

FIGS. 20A-20C: Alignment of the G proteins of different isolates of hMPVof all four variants, variant A1, A2, B1, or B2 (SEQ ID NOS:119-153).

FIG. 21: Phylogenetic tree based on the F gene sequences showing thephylogenetic relationship of the different hMPV isolates with therespective variants of hMPV.

FIG. 22: Phylogenetic tree based on the G gene sequences showing thephylogenetic relationship of the different hMPV isolates with therespective variants of hMPV is shown in FIG. 13.

FIG. 23: Growth curve of hMPV isolate 00-1 (A1) in Vero cells. The Verocells were infected at a MOI of 0.1.

FIGS. 24A-24B: Sequence of CAT-hMPV minireplicon construct (SEQ IDNOS:482-484). The function encoded by a segment of sequence is indicatedunderneath the sequence.

FIG. 25: Expression of CAT from the CAT-hMPV minireplicon. The differentconstructs used for transfection are indicated on the x-axis; the amountof CAT expression is indicated on the y-axis. The Figure shows CATexpression 24 hours after transfection and CAT expression 48 hours aftertransfection. Standards were dilutions of CAT protein.

FIG. 26: Leader and Trailer Sequence Comparison: Alignments of theleader and trailer sequences of different viruses as indicated are shown(SEQ ID NOS:485-496).

FIG. 27: hMPV genome analysis: PCR fragments of hMPV genomic sequencerelative to the hMPV genomic organization are shown. The position ofmutations are shown underneath the vertical bars indicating the PCRfragments.

FIG. 28: Restriction maps of hMPV isolate 00-1 (A1) and hMPV isolate99-1 (B1). Restriction sites in the respective isolates are indicatedunderneath the diagram showing the genomic organization of hMPV. Thescale on top of the diagram indicates the position in the hMPV genome inkb.

FIGS. 29A and 29B: hMPV cDNA assembly. The diagram on top shows thegenomic organization of hMPV, the bars underneath indicate the PCRfragments (see FIG. 27) that are assembled to result in a full lengthcDNA encoding the virus. The numbers on top of the bars representing thePCR fragments indicate the position in the viral genome in base pairs.

FIGS. 30A-30C: Nucleotide (SEQ ID NO:497) and amino acid (SEQ ID NO:498)sequence information from the 3′ end of the genome of MPV isolate 00-1(A1). ORFs are given. N: ORF for nucleoprotein; P: ORF forphosphoprotein; M: ORF for matrix protein; F: ORF for fusion protein;GE: gene end; GS: gene start.

FIGS. 31A and B: Nucleotide (SEQ ID NOS:499 and 501) and amino acid (SEQID NOS:500 and 502) sequence information from obtained fragments in thepolymerase gene (L) of MPV isolates 00-1 (A1). Positioning of thefragments in L is based on protein homologies with APV-A (accessionnumber U65312). The translated fragment 8 (FIG. 31A) is located at aminoacid number 8 to 243, and the consensus of fragments 9 and 10 (FIG. 31B)is located at amino acid number 1358 to 1464 of the APV-A L ORF.

FIG. 32: Results of RT-PCR assays on throat and nose swabs of 12 guineapigs 15 inoculated with ned/00/01 (A1) and/or ned/99/01 (B1).

FIG. 33A: IgG response against ned/00/01 (A1) and ned/99/01 (B1) forguinea pigs infected with ned/00/01 (A1) and re-infected with ned//00/01(A1) (GP 4, 5 and 6) or ned/99/01 (B1) (GP 1 and 3).

FIG. 33B: IgG response against ned/00/01 (A1) and ned/99/01 (B1) forguinea pigs infected with ned/99/01 and re-infected with eitherned/00/01 (A1) (GP's 8 and 9) or with ned/99/01 (B1) (GP's 10, 11, 12).

FIG. 34: Specificity of the ned/00/01 (A1) and ned/99/01 (B1) ELISA onsera taken from guinea pigs infected with either ned/00/01 (A1) orned/99/01 (B1).

FIG. 35: Mean IgG response against ned/00/01 (A1) and ned/99/01 (B1)ELISA of 3 homologous (00-1/00-1), 2 homologous (99-1/99-1), 2heterologous (99-1/00-1) and 2 heterologous (00-1/99-1) infected guineapigs.

FIG. 36: Mean percentage of APV inhibition of hMPV infected guinea pigs.

FIG. 37: Virus neutralization titers of ned/00/01 (A1) and ned/99/01(B1) infected guinea pigs against ned/00/01 (A1), ned/99/01 (B1) andAPV-C.

FIG. 38: Results of RT-PCR assays on throat swabs of cynomolgousmacaques inoculated (twice) with ned/00/01 (A1).

FIG. 39A (top two panels): IgA, IgM and IgG response against ned/00/01(A1) of 2 cynomologous macaques (re)infected with ned/00/01 (A1).

FIG. 39B (bottom panels): IgG response against APV of 2 Cynomologousmacaques infected with ned/00/01 (A1).

FIG. 40: Comparison of the use of the hMPV ELISA and the APV inhibitionELISA for the detection of IgG antibodies in human sera.

FIG. 41: Comparison of two prototypic hMPV isolates with APV-A andAPV-C; DNA similarity matrices for nucleic acids encoding the variousviral proteins.

FIG. 42A: Comparison of two prototypic hMPV isolates with APV-A andAPV-C; protein similarity matrices for the various viral proteins.

FIG. 42B: Comparison of the coding sequences of four prototypes ofmammalian MPV. The left column shows nucleic acid sequence comparisonsand the right column shows amino acid sequence comparisons. NL/1/00 isthe prototype of variant A1 (SEQ ID NO:19). NL/17/00 is the prototype ofvariant A2 (SEQ ID NO:20). NL/1/99 the prototype of variant B1 (SEQ IDNO:18). NL/1/94 is the prototype of variant B2 (SEQ ID NO:21).

FIG. 43: Amino acid alignment of the nucleoprotein of two prototype hMPVisolates (SEQ ID NOS:503 and 504).

FIG. 44: Amino acid alignment of the phosphoprotein of two prototypehMPV isolates (SEQ ID NOS:505 and 506).

FIG. 45: Amino acid alignment of the matrix protein of two prototypehMPV isolates (SEQ ID NOS:507 and 508).

FIG. 46: Amino acid alignment of the fusion protein of two prototypehMPV isolates (SEQ ID NOS:509 and 510).

FIG. 47: Amino acid alignment of the M2-1 protein of two prototype hMPVisolates (SEQ ID NOS:511 and 512).

FIG. 48: Amino acid alignment of the M2-2 protein of two prototype hMPVisolates (SEQ ID NOS:513 and 514).

FIG. 49: Amino acid alignment of the short hydrophobic protein of twoprototype hMPV isolates (SEQ ID NOS:515 and 516).

FIG. 50: Amino acid alignment of the attachment glycoprotein of twoprototype hMPV isolates (SEQ ID NOS:517 and 518).

FIG. 51: Amino acid alignment of the N-terminus of the polymeraseprotein of two prototype hMPV isolates (SEQ ID NOS:519 and 520).

FIG. 52: Noncoding sequences of hMPV isolate 00-1 (A1). (A) Thenoncoding sequences (SEQ ID NOS:521-529) between the ORFs and at thegenomic termini are shown in the positive sense. From left to right,stop codons of indicated ORFs are shown, followed by the noncodingsequences, the gene start signals and start codons of the indicatedsubsequent ORFs. Numbers indicate the first position of start and stopcodons in the hMPV map. Sequences that display similarity to publishedgene end signals are underlined and sequences that display similarity toUAAAAAU/A/C are represented with a line above the sequence. (B)Nucleotide sequences (SEQ ID NOS:530-535) of the genomic termini ofhMPV. The genomic termini of hMPV are aligned with each other and withthose of APV. Underlined regions represent the primer sequences used inRT-PCR assays which are based on the 3′ and 5′ end sequences of APV andRSV. Bold italicized nucleotides are part of the gene start signal ofthe N gene. Le: leader, Tr: trailer.

FIGS. 53A-53JJ: Sequence comparison of the genomic sequence of hMPVisolate 00-1 (A1) with hMPV isolate 99-1 (B1) (SEQ ID NOS:536-539).

FIG. 54: Leader sequences of human metapneumovirus (hMPV) NL/1/00 (A1)genomic RNA was determined using a combination of polyadenylation and 3′RACE methods.

FIG. 55: Sequencing analyses on PCR products directly and on PCR clonesboth indicated that the leader region of hMPV consisted of 5′ ACG CGAAAA AAA CGC GTA TA (expressed as positive sense cDNA orientation) at the3′ most proximal 20 nucleotides in the leader sequence (SEQ IDNOS:540-543). The two newly identified nucleotides are underlined.

5. DETAILED DESCRIPTION OF THE INVENTION

The invention relates to an isolated mammalian negative strand RNAvirus, metapneumovirus (MPV) and variants thereof, within the sub-familyPneumovirinae, of the family Paramyxoviridae. The present invention alsorelates to isolated mammalian negative strand RNA viruses identifiableas phylogenetically corresponding or relating to the genusmetapneumovirus and components thereof. The mammalian MPVs of theinvention can be a variant A1, A2, B1 or B2 mammalian MPV. However, themammalian MPVs of the present invention may encompass additionalvariants of MPV yet to be identified, and are not limited to variantsA1, A2, B1 or B2.

The invention relates to genomic nucleotide sequences of differentvariants of isolates of mammalian metapneumoviruses (MPV), in particularhuman metapneumoviruses including isolates of variants A1, A2, B1 andB2. The invention relates to the use of the sequence information ofdifferent isolates of mammalian metapneumoviruses for diagnostic andtherapeutic methods. The present invention relates to the differences ofthe genomic nucleotide sequences among the differentmetapneumovirus-isolates, and their use in the diagnostic andtherapeutic methods of the invention. In particular, the inventionrelates to the use of the single nucleotide polymorphisms (SNPs) amongdifferent metapneumovirus isolates for diagnostic and therapeuticmethods. The present invention also relates to the use serologicalcharacterization of the different isolates of mammalianmetapneumoviruses, alone or in combination with the sequence informationof the different isolates, for diagnostic and therapeutic methods.

The present invention relates to nucleotide sequences encoding thegenome of a metapneumovirus or a portion thereof, including bothmammalian and avian metapneumovirus (APV). The present invention relatesto nucleotide sequences encoding gene products of a metapneumovirus,including both mammalian and avian metapneumoviruses. The presentinvention further relates to nucleic acids, including DNA and RNA, thatencode the genome or a portion thereof of a metapneumovirus, includingboth mammalian and avian, in addition to a nucleotide sequence, which isheterologous or non-native to the viral genome. The invention furtherencompasses recombinant or chimeric viruses encoded by the nucleotidesequences.

In accordance with the present invention, a recombinant virus is onederived from a mammalian MPV or an APV that is encoded by endogenous ornative genomic sequences or non-native genomic sequences. In accordancewith the invention, a non-native sequence is one that is different fromthe native or endogenous genomic sequence due to one or more mutations,including, but not limited to, point mutations, rearrangements,insertions, deletions etc., the genomic sequence that may or may notresult in a phenotypic change. In accordance with the invention, achimeric virus is a recombinant MPV or APV which further comprises aheterologous nucleotide sequence. In accordance with the invention, achimeric virus may be encoded by a nucleotide sequence in whichheterologous nucleotide sequences have been added to the genome or inwhich endogenous or native nucleotide sequences have been replaced withheterologous nucleotide sequences.

The invention further relates to vaccine formulations comprisingmammalian or avian metapneumovirus, including recombinant forms of theviruses. In particular, the present invention encompasses vaccinepreparations comprising recombinant or chimeric forms of MPV or APV thatexpress antigenic glycoproteins, including glycoproteins of MPV, or APVand/or non-native MPV or APV glycoproteins. The invention alsoencompasses vaccine preparations comprising recombinant forms of MPV orAPV that encode antigenic sequences of another negative strand RNAvirus, including PTV or RSV, or a heterologous glycoprotein of anotherspecies or strain of metapneumovirus. The invention further relates tovaccines comprising chimeric hMPV wherein the chimeric hMPV encodes oneor more APV proteins and wherein the chimeric hMPV optionallyadditionally expresses one or more heterologous or non-native sequences.The invention also relates to vaccines comprising chimeric APV whereinthe chimeric APV encodes one or more hMPV proteins and wherein thechimeric APV optionally additionally expresses one or more heterologousor non-native sequences. The present invention also relates tomultivalent vaccines, including bivalent and trivalent vaccines. Inparticular, the bivalent and trivalent vaccines of the inventionencompass two or more antigenic polypeptides expressed by the same ordifferent pneumoviral vectors encoding antigenic proteins of MPV, APV,PIV, RSV, influenza or another negative strand RNA virus, ormorbillivirus.

5.1 Mammalian Metapneumovirus Structural Characteristics of a MammalianMetapneumovirus

The invention provides a mammalian MPV. The mammalian MPV is anegative-sense single-stranded RNA virus belonging to the sub-familyPneumovirinae of the family Paramyxoviridae. Moreover, the mammalian MPVis identifiable as phylogenetically corresponding to the genusMetapneumovirus, wherein the mammalian MPV is phylogenetically moreclosely related to a virus isolate deposited as 1-2614 with CNCM, Paris(SEQ ID NO:19) than to turkey rhinotracheitis virus, the etiologicalagent of avian rhinotracheitis. A virus is identifiable asphylogenetically corresponding to the genus Metapneumovirus by, e.g.,obtaining nucleic acid sequence information of the virus and testing itin phylogenetic analyses. Any technique known to the skilled artisan canbe used to determine phylogenetic relationships between strains ofviruses. For exemplary methods see section 5.9. Other techniques aredisclosed in International Patent Application PCT/NL02/00040, publishedas WO 02/057302, which is incorporated by reference in its entiretyherein. In particular, PCT/NL02/00040 discloses nucleic acid sequencesthat are suitable for phylogenetic analysis at page 12, line 27 to page19, line 29, which are incorporated by reference herein. A virus canfurther be identified as a mammalian MPV on the basis of sequencesimilarity as described in more detail below.

In addition to phylogenetic relatedness and sequence similarity of avirus to a mammalian MPV as disclosed herein, the similarity of thegenomic organization of a virus to the genomic organization of amammalian MPV disclosed herein can also be used to identify the virus asa mammalian MPV. For a representative genomic organization of amammalian MPV see FIG. 27. In certain embodiments, the genomicorganization of a mammalian MPV is different from the genomicorganization of pneumoviruses within the sub-family Pneumovirinae of thefamily Paramyxoviridae. The classification of the two genera,metapneumovirus and pneumovirus, is based primarily on their geneconstellation; metapneumoviruses generally lack non-structural proteinssuch as NS1 or NS2 (see also Randhawa et al., 1997, J. Virol.71:9849-9854) and the gene order is different from that of pneumoviruses(RSV: ‘3-NS1-NS2-N-P-M-5H-G-F-M2-L-5’, APV: ‘3-N-P-M-F-M2-5H-G-L-5’)(Lung, et al., 1992, J. Gen. Virol. 73:1709-17 15; Yu, et al., 1992,Virology 186:426-434; Randhawa, et al., 1997, J. Virol. 71:9849-9854).

Further, a mammalian MPV of the invention can be identified by itsimmunological properties. In certain embodiments, specific anti-sera canbe raised against mammalian MPV that can neutralize mammalian MPV.Monoclonal and polyclonal antibodies can be raised against MPV that canalso neutralize mammalian MPV. (See, PCT WO 02/057302 at pages 36 to 97,which is incorporated by reference herein.

The mammalian MPV of the invention is further characterized by itsability to infect a mammalian host, i.e., a mammalian cultured cell or amammal. Unlike APV, mammalian MPV does not replicate or replicates onlyat low levels in chickens and turkeys. Mammalian MPV replicates,however, in mammalian hosts, such as cynomolgous macaques. In certain,more specific, embodiments, a mammalian MPV is further characterized byits ability to replicate in a mammalian host. In certain, more specificembodiments, a mammalian MPV is further characterized by its ability tocause the mammalian host to express proteins encoded by the genome ofthe mammalian MPV. In even more specific embodiments, the viral proteinsexpressed by the mammalian MPV are inserted into the cytoplasmicmembranes of the mammalian host. In certain embodiments, the mammalianMPV of the invention can infect a mammalian host and cause the mammalianhost to produce new infectious viral particles of the mammalian MPV. Fora more detailed description of the functional characteristics of themammalian MPV of the invention, see section 5.1.2.

In certain embodiments, the appearance of a virus in an electronmicroscope or its sensitivity to chloroform can be used to identify thevirus as a mammalian MPV. The mammalian MPV of the invention appears inan electron microscope as paramyxovirus-like particle. Consistently, amammalian MPV is sensitive to treatment with chloroform; a mammalian MPVis cultured optimally on tMK cells or cells functionally equivalentthereto and it is essentially trypsine dependent in most cell cultures.Furthermore, a mammalian MPV has a typical cytopathic effects (CPE) andlacks hemagglutinating activity against species of red blood cells. TheCPE induced by MPV isolates are similar to the CPE induced by hRSV, withcharacteristic syncytia formation followed by rapid internal disruptionof the cells and subsequent detachment from the culture plates. Althoughmost paramyxoviruses have hemagglutinating activity, most of thepneumoviruses do not (Pringle, C. R. In: The Paramyxoviruses; (ed. D. W.Kingsbury) 1-39 (Plenum Press, New York, 1991)). A mammalian MPVcontains a second overlapping ORF (M2-2) in the nucleic acid fragmentencoding the M2 protein. The occurrence of this second overlapping ORFoccurs in other pneumoviruses as shown in Ahmadian et al., 1999, J. Gen.Vir. 80:2011-2016.

In certain embodiments, the invention provides methods to identify aviral isolate as a mammalian MPV. A test sample can, e.g., be obtainedfrom an animal or human. The sample is then tested for the presence of avirus of the sub-family Pneumovirinae. If a virus of the sub-familyPneumovirinae is present, the virus can be tested for any of thecharacteristics of a mammalian MPV as discussed herein, such as, but notlimited to, phylogenetic relatedness to a mammalian MPV, nucleotidesequence identity to a nucleotide sequence of a mammalian MPV, aminoacid sequence identity/homology to an amino acid sequence of a mammalianMPV, and genomic organization. Furthermore, the virus can be identifiedas a mammalian MPV by cross-hybridization experiments using nucleic acidsequences from a MPV isolate, RT-PCR using primers specific to mammalianMPV, or in classical cross-serology experiments using antibodiesdirected against a mammalian MPV isolate. In certain other embodiments,a mammalian MPV can be identified on the basis of its immunologicaldistinctiveness, as determined by quantitative neutralization withanimal antisera. The antisera can be obtained from, e.g., ferrets, pigsor macaques that are infected with a mammalian MPV (see, e.g., Example8).

In certain embodiments, the serotype does not cross-react with virusesother than mammalian MPV. In other embodiments, the serotype shows ahomologous-to-heterologous titer ratio >16 in both directions Ifneutralization shows a certain degree of cross-reaction between twoviruses in either or both directions (homologous-to-heterologous titerration of eight or sixteen), distinctiveness of serotype is assumed ifsubstantial biophysical/biochemical differences of DNA sequences exist.If neutralization shows a distinct degree of cross-reaction between twoviruses in either or both directions (homologous-to-heterologous titerratio of smaller than eight), identity of serotype of the isolates understudy is assumed. Isolate I-2614, herein also known as MPV isolate 00-1,can be used as prototype.

In certain embodiments, a virus can be identified as a mammalian MPV bymeans of sequence homology/identity of the viral proteins or nucleicacids in comparison with the amino acid sequence and nucleotidesequences of the viral isolates disclosed herein by sequence or deposit.In particular, a virus is identified as a mammalian MPV when the genomeof the virus contains a nucleic acid sequence that has a percentagenucleic acid identity to a virus isolate deposited as I-2614 with CNCM,Paris which is higher than the percentages identified herein for thenucleic acids encoding the L protein, the M protein, the N protein, theP protein, or the F protein as identified herein below in comparisonwith APV-C (see Table 1). (See, PCT WO 02/05302, at pp. 12 to 19, whichis incorporated by reference herein. Without being bound by theory, itis generally known that viral species, especially RNA virus species,often constitute a quasi-species wherein the members of a cluster of theviruses display sequence heterogeneity. Thus, it is expected that eachindividual isolate may have a somewhat different percentage of sequenceidentity when compared to APV-C.

The highest amino sequence identity between the proteins of MPV and anyof the known other viruses of the same family to date is the identitybetween APV-C and human MPV. Between human MPV and APV-C, the amino acidsequence identity for the matrix protein is 87%, 88% for thenucleoprotein, 68% for the phosphoprotein, 81% for the fusion proteinand 56-64% for parts of the polymerase protein, as can be deduced whencomparing the sequences given in FIGS. 30A-30C, see also Table 1. Viralisolates that contain ORFs that encode proteins with higher homologycompared to these maximum values are considered mammalian MPVs. Itshould be noted that, similar to other viruses, a certain degree ofvariation is found between different isolated of mammalian MPVs.

TABLE 1 Amino acid sequence identity between the ORFs of MPV and thoseof other paramyxoviruses. N P M F M2-1 M2-2 L APV A 69 55 78 67 72 26 64APV B 69 51 76 67 71 27 ⁻² APV C 88 68 87 81 84 56 ⁻² hRSV A 42 24 38 3436 18 42 hRSV B 41 23 37 33 35 19 44 bRSV 42 22 38 34 35 13 44 PVM 45 2637 39 33 12 ⁻² others³ 7-11 4-9 7-10 10-18 ⁻⁴ ⁻⁴ 13-14 Footnotes: 1. Nosequence homologies were found with known G and SH proteins and werethus excluded. ²Sequences not available. ³others: human parainfluenzavirus type 2 and 3, Sendai virus, measles virus, nipah virus, phocinedistemper virus, and New Castle Disease virus. ⁴ORF absent in viralgenome.

In certain embodiments, the invention provides a mammalian MPV, whereinthe amino acid sequence of the SH protein of the mammalian MPV is atleast 30%, at least 35%, at least 40%, at least 45%, at least 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 98%, atleast 99%, or at least 99.5% identical to the amino acid sequence of SEQID NO:382 (SH protein of isolate NL/1/00; see Table 14). The isolatednegative-sense single-stranded RNA metapneumovirus that comprises the SHprotein that is at least 30% identical to SEQ ID NO:382 (SH protein ofisolate NL/1/00; see Table 14) is capable, of infecting a mammalianhost. In certain embodiments, the isolated negative-sensesingle-stranded RNA metapneumovirus that comprises the SH protein thatis at least 30% identical to SEQ ID NO:382 (SH protein of isolateNL/1/00; see Table 14) is capable of replicating in a mammalian host. Incertain embodiments, a mammalian MPV contains a nucleotide sequence thatencodes a SH protein that is at least 30% identical to SEQ ID NO:382 (SHprotein of isolate NL/1/00; see Table 14).

In certain embodiments, the invention provides a mammalian MPV, whereinthe amino acid sequence of the G protein of the mammalian MPV is atleast 20%, at least 25%, at least 30%, at least 35%, at least 40%, atleast 45%, at least 50%, at least 55%, at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 98%, at least 99%, or at least 99.5% identical tothe amino acid sequence of SEQ ID NO:322 (G protein of isolate NL/1/00;see Table 14). The isolated negative-sense single-stranded RNAmetapneumovirus that comprises the G protein that is at least 20%identical to SEQ ID NO:322 (G protein of isolate NL/1/00; see Table 14)is capable of infecting a mammalian host. In certain embodiments, theisolated negative-sense single-stranded RNA metapneumovirus thatcomprises the G protein that is at least 20% identical to SEQ ID NO:322(G protein of isolate NL/1/00; see Table 14) is capable of replicatingin a mammalian host. In certain embodiments, a mammalian MPV contains anucleotide sequence that encodes a G protein that is at least 20%identical to SEQ ID NO:322 (G protein of isolate NL/1/00; see Table 14).

In certain embodiments, the invention provides a mammalian MPV, whereinthe amino acid sequence of the L protein of the mammalian MPV is atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or atleast 99.5% identical to the amino acid sequence of SEQ ID NO:330 (Lprotein of isolate NL/1/00; see Table 14). The isolated negative-sensesingle-stranded RNA metapneumovirus that comprises the L protein that isat least 85% identical to SEQ ID NO:330 (L protein of isolate NL/1/00;see Table 14) is capable of infecting a mammalian host. In certainembodiments, the isolated negative-sense single-stranded RNAmetapneumovirus that comprises the L protein that is at least 85%identical to SEQ ID NO:330 (L protein of isolate NL/1/00; see Table 14)is capable of replicating in a mammalian host. In certain embodiments, amammalian MPV contains a nucleotide sequence that encodes a L proteinthat is at least 20% identical to SEQ ID NO:330 (L protein of isolateNL/1/00; see Table 14).

In certain embodiments, the invention provides a mammalian MPV, whereinthe amino acid sequence of the N protein of the mammalian MPV is atleast 90%, at least 95%, or at least 98% identical to the amino acidsequence of SEQ ID NO:366. The isolated negative-sense single-strandedRNA metapneumovirus that comprises the N protein that is at least 90%identical in amino acid sequence to SEQ ID NO:366 is capable ofinfecting mammalian host. In certain embodiments, the isolatednegative-sense single-stranded RNA metapneumovirus that comprises the Nprotein that is 90% identical in amino acid sequence to SEQ ID NO:366 iscapable of replicating in a mammalian host. The amino acid identity iscalculated over the entire length of the N protein. In certainembodiments, a mammalian MPV contains a nucleotide sequence that encodesa N protein that is at least 90%, at least 95%, or at least 98%identical to the amino acid sequence of SEQ ID NO:366.

The invention further provides mammalian MPV, wherein the amino acidsequence of the P protein of the mammalian MPV is at least 70%, at least80%, at least 90%, at least 95% or at least 98% identical to the aminoacid sequence of SEQ ID NO:374. The mammalian MPV that comprises the Pprotein that is at least 70% identical in amino acid sequence to SEQ IDNO:374 is capable of infecting a mammalian host. In certain embodiments,the mammalian MPV that comprises the P protein that is at least 70%identical in amino acid sequence to SEQ ID NO:374 is capable ofreplicating in a mammalian host. The amino acid identity is calculatedover the entire length of the P protein. In certain embodiments, amammalian MPV contains a nucleotide sequence that encodes a P proteinthat is at least 70%, at least 80%, at least 90%, at least 95% or atleast 98% identical to the amino acid sequence of SEQ ID NO:374.

The invention further provides, mammalian MPV, wherein the amino acidsequence of the M protein of the mammalian MPV is at least 90%, at least95% or at least 98% identical to the amino acid sequence of SEQ IDNO:358. The mammalian MPV that comprises the M protein that is at least90% identical in amino acid sequence to SEQ ID NO:358 is capable ofinfecting mammalian host. In certain embodiments, the isolatednegative-sense single-stranded RNA metapneumovirus that comprises the Mprotein that is 90% identical in amino acid sequence to SEQ ID NO:358 iscapable of replicating in a mammalian host. The amino acid identity iscalculated over the entire length of the M protein. In certainembodiments, a mammalian MPV contains a nucleotide sequence that encodesa M protein that is at least 90%, at least 95% or at least 98% identicalto the amino acid sequence of SEQ ID NO:358.

The invention further provides mammalian MPV, wherein the amino acidsequence of the F protein of the mammalian MPV is at least 85%, at least90%, at least 95% or at least 98% identical to the amino acid sequenceof SEQ ID NO:314. The mammalian MPV that comprises the F protein that isat least 85% identical in amino acid sequence to SEQ ID NO:314 iscapable of infecting a mammalian host. In certain embodiments, theisolated negative-sense single-stranded RNA metapneumovirus thatcomprises the F protein that is 85% identical in amino acid sequence toSEQ ID NO:314 is capable of replicating in mammalian host. The aminoacid identity is calculated over the entire length of the F protein. Incertain embodiments, a mammalian MPV contains a nucleotide sequence thatencodes a F protein that is at least 85%, at least 90%, at least 95% orat least 98% identical to the amino acid sequence of SEQ ID NO:314.

The invention further provides mammalian MPV, wherein the amino acidsequence of the M2-1 protein of the mammalian MPV is at least 85%, atleast 90%, at least 95% or at least 98% identical to the amino acidsequence of SEQ ID NO:338. The mammalian MPV that comprises the M2-1protein that is at least 85% identical in amino acid sequence to SEQ IDNO:338 is capable of infecting a mammalian host. In certain embodiments,the isolated negative-sense single-stranded RNA metapneumovirus thatcomprises the M2-1 protein that is 85% identical in amino acid sequenceto SEQ ID NO:338 is capable of replicating in a mammalian host. Theamino acid identity is calculated over the entire length of the M2-1protein. In certain embodiments, a mammalian MPV contains a nucleotidesequence that encodes a M2-1 protein that is at least 85%, at least 90%,at least 95% or at least 98% identical to the amino acid sequence of SEQID NO:338.

The invention further provides mammalian MPV, wherein the amino acidsequence of the M2-2 protein of the mammalian MPV is at least 60%, atleast 70%, at least 80%, at least 90%, at least 95% or at least 98%identical to the amino acid sequence of SEQ ID NO:346 The isolatedmammalian MPV that comprises the M2-2 protein that is at least 60%identical in amino acid sequence to SEQ ID NO:346 is capable ofinfecting mammalian host. In certain embodiments, the isolatednegative-sense single-stranded RNA metapneumovirus that comprises theM2-2 protein that is 60% identical in amino acid sequence to SEQ IDNO:346 is capable of replicating in a mammalian host. The amino acididentity is calculated over the entire length of the M2-2 protein. Incertain embodiments, a mammalian MPV contains a nucleotide sequence thatencodes a M2-1 protein that is at least 60%, at least 70%, at least 80%,at least 90%, at least 95% or at least 98% identical to the amino acidsequence of SEQ ID NO:346.

In certain embodiments, the invention provides mammalian MPV, whereinthe negative-sense single-stranded RNA metapneumovirus encodes at leasttwo proteins, at least three proteins, at least four proteins, at leastfive proteins, or six proteins selected from the group consisting of (i)a N protein with at least 90% amino acid sequence identity to SEQ IDNO:366; (ii) a P protein with at least 70% amino acid sequence identityto SEQ ID NO:374 (iii) a M protein with at least 90% amino acid sequenceidentity to SEQ ID NO:358 (iv) a F protein with at least 85% amino acidsequence identity to SEQ ID NO:314 (v) a M2-1 protein with at least 85%amino acid sequence identity to SEQ ID NO:338; and (vi) a M2-2 proteinwith at least 60% amino acid sequence identity to SEQ ID NO:346.

The invention provides two subgroups of mammalian MPV, subgroup A andsubgroup B. The invention also provides four variants A1, A2, B1 and B2.A mammalian MPV can be identified as a member of subgroup A if it isphylogenetically closer related to the isolate 00-1 (SEQ ID NO:19) thanto the isolate 99-1 (SEQ ID NO:18). A mammalian MPV can be identified asa member of subgroup B if it is phylogenetically closer related to theisolate 99-1 (SEQ ID NO:18) than to the isolate 00-1 (SEQ ID NO:19). Inother embodiments, nucleotide or amino acid sequence homologies ofindividual ORFS can be used to classify a mammalian MPV as belonging tosubgroup A or B.

The different isolates of mammalian MPV can be divided into fourdifferent variants, variant A1, variant A2, variant 81 and variant B2(see FIGS. 21 and 22). The isolate 00-1 (SEQ ID NO:19) is an example ofthe variant A1 of mammalian MPV. The isolate 99-1 (SEQ ID NO:18) is anexample of the variant B1 of mammalian MPV. A mammalian MPV can begrouped into one of the four variants using a phylogenetic analysis.Thus, a mammalian MPV belongs to a specific variant if it isphylogenetically closer related to a known member of that variant thanit is phylogenetically related to a member of another variant ofmammalian MPV. The sequence of any ORF and the encoded polypeptide maybe used to type a MPV isolate as belonging to a particular subgroup orvariant, including N, P, L, M, SH, G, M2 or F polypeptides. In aspecific embodiment, the classification of a mammalian MPV into avariant is based on the sequence of the G protein. Without being boundby theory, the G protein sequence is well suited for phylogeneticanalysis because of the high degree of variation among G proteins of thedifferent variants of mammalian MPV.

In certain embodiments of the invention, sequence homology may bedetermined by the ability of two sequences to hybridize under certainconditions, as set forth below. A nucleic acid which is hybridizable toa nucleic acid of a mammalian MPV, or to its reverse complement, or toits complement can be used in the methods of the invention to determinetheir sequence homology and identities to each other. In certainembodiments, the nucleic acids are hybridized under conditions of highstringency.

It is well-known to the skilled artisan that hybridization conditions,such as, but not limited to, temperature, salt concentration, pH,formamide concentration (see, e.g., Sambrook et al., 1989, Chapters 9 to11, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., incorporated herein byreference in its entirety). In certain embodiments, hybridization isperformed in aqueous solution and the ionic strength of the solution iskept constant while the hybridization temperature is varied dependent onthe degree of sequence homology between the sequences that are to behybridized. For DNA sequences that 100% identical to each other and arelonger than 200 base pairs, hybridization is carried out atapproximately 15-25° C. below the melting temperature (Tm) of theperfect hybrid. The melting temperature (Tm) can be calculated using thefollowing equation (Bolton and McCarthy, 1962, Proc. Natl. Acad. Sci.USA 84:1390):Tm=81.5° C.−16.6(log 10[Na+])+(% G+C)−0.63(% formamide)−(600/l)Wherein (Tm) is the melting temperature, [Na+] is the sodiumconcentration, G+C is the Guanine and Cytosine content, and 1 is thelength of the hybrid in basepairs. The effect of mismatches between thesequences can be calculated using the formula by Bonner et al. (Bonneret al., 1973, J. Mol. Biol. 81:123-135): for every 1% of mismatching ofbases in the hybrid, the melting temperature is reduced by 1-1.5° C.

Thus, by determining the temperature at which two sequences hybridize,one of skill in the art can estimate how similar a sequence is to aknown sequence. This can be done, e.g., by comparison of the empiricallydetermined hybridization temperature with the hybridization temperaturecalculated for the know sequence to hybridize with its perfect match.Through the use of the formula by Bonner et al., the relationshipbetween hybridization temperature and percent mismatch can be exploitedto provide information about sequence similarity.

By way of example and not limitation, procedures using such conditionsof high stringency are as follows. Prehybridization of filterscontaining DNA is carried out for 8 hours to overnight at 65° C. inbuffer composed of 6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP,0.02% Ficoll, 0.02% BSA, and 500 μg/ml denatured salmon sperm DNA.Filters are hybridized for 48 hours at 65° C. in prehybridizationmixture containing 100 μg/ml denatured salmon sperm DNA and 5-20×10⁶ cpmof 32P-labeled probe. Washing of filters is done at 37° C. for 1 hour ina solution containing 2×SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA.This is followed by a wash in 0.1×SSC at 50° C. for 45 minutes beforeautoradiography. Other conditions of high stringency which may be usedare well known in the art. In other embodiments of the invention,hybridization is performed under moderate of low stringency conditions,such conditions are well-known to the skilled artisan (see e.g.,Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2d Ed.,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; see also,Ausubel et al., eds., in the Current Protocols in Molecular Biologyseries of laboratory technique manuals, 1987-1997 Current Protocols,©1994-1997 John Wiley and Sons, Inc., each of which is incorporated byreference herein in their entirety). An illustrative low stringencycondition is provided by the following system of buffers: hybridizationin a buffer comprising 35% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg/ml denatured salmonsperm DNA, and 10% (wt/vol) dextran sulfate for 18-20 hours at 40° C.,washing in a buffer consisting of 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mMEDTA, and 0.1% SDS for 1.5 hours at 55° C., and washing in a bufferconsisting of 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDSfor 1.5 hours at 60° C.

In certain embodiments, a mammalian MPV can be classified into one ofthe variant using probes that are specific for a specific variant ofmammalian MPV. Such probes include primers for RT-PCR and antibodies.Illustrative methods for identifying a mammalian MPV as a member of aspecific variant are described in section 5.9 below.

In certain embodiments of the invention, the different variants ofmammalian MPV can be distinguished from each other by way of the aminoacid sequences of the different viral proteins (see, e.g., FIG. 42B). Inother embodiments, the different variants of mammalian MPV can bedistinguished from each other by way of the nucleotide sequences of thedifferent ORFs encoded by the viral genome (see, e.g., FIG. 42B). Avariant of mammalian MPV can be, but is not limited to, A1, A2, B1 orB2. The invention, however, also contemplates isolates of mammalian MPVthat are members of another variant yet to be identified. The inventionalso contemplates that a virus may have one or more ORF that are closerrelated to one variant and one or more ORFs that are closerphylogenetically related to another variant. Such a virus would beclassified into the variant to which the majority of its ORFs are closerphylogenetically related. Non-coding sequences may also be used todetermine phylogenetic relatedness.

An isolate of mammalian MPV is classified as a variant B1 if it isphylogenetically closer related to the viral isolate NL/1199 (SEQ IDNO:18) than it is related to any of the following other viral isolates:NL/1/00 (SEQ ID NO:19), NL/17/00 (SEQ ID NO:20) and NL/1/94 (SEQ IDNO:21). One or more of the ORFs of a mammalian MPV can be used toclassify the mammalian MPV into a variant. A mammalian MPV can beclassified as an MPV variant B1, if the amino acid sequence of its Gprotein is at least 66%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, or at least 99% orat least 99.5% identical to the G protein of a mammalian MPV variant B1as represented by the prototype NL/1/99 (SEQ ID NO:324); if the aminoacid sequence of its N protein is at least 98.5% or at least 99% or atleast 99.5% identical to the N protein of a mammalian MPV variant B1 asrepresented by the prototype NL/1/99 (SEQ ID NO:368); if the amino acidsequence of its P protein is at least 96%, at least 98%, or at least 99%or at least 99.5% identical to the P protein of a mammalian MPV variantB1 as represented by the prototype NL/1/99 (SEQ ID NO:376); if the aminoacid sequence of its M protein is identical to the M protein of amammalian MPV variant B1 as represented by the prototype NL/1/99 (SEQ IDNO:360); if the amino acid sequence of its F protein is at least 99%identical to the F protein of a mammalian MPV variant B1 as representedby the prototype NL/1/99 (SEQ ID NO:316); if the amino acid sequence ofits M2-1 protein is at least 98% or at least 99% or at least 99.5%identical to the M2-1 protein of a mammalian MPV variant B1 asrepresented by the prototype NL/1/99 (SEQ ID NO:340); if the amino acidsequence of its M2-2 protein is at least 99% or at least 99.5% identicalto the M2-2 protein of a mammalian MPV variant B1 as represented by theprototype NL/1/99 (SEQ ID NO:348); if the amino acid sequence of its SHprotein is at least 83%, at least 85%, at least 90%, at least 95%, atleast 98%, or at least 99% or at least 99.5% identical to the SH proteinof a mammalian MPV variant B1 as represented by the prototype NL/1/99(SEQ ID NO:384); and/or if the amino acid sequence of its L protein isat least 99% or at least 99.5% identical to the L protein a mammalianMPV variant B1 as represented by the prototype NL/1/99 (SEQ ID NO:332).

An isolate of mammalian MPV is classified as a variant A1 if it isphylogenetically closer related to the viral isolate NL/1/00 (SEQ IDNO:19) than it is related to any of the following other viral isolates:NL/1/99 (SEQ ID NO:18), NL/17/00 (SEQ ID NO:20) and NL/1/94 (SEQ IDNO:21). One or more of the ORFs of a mammalian MPV can be used toclassify the mammalian MPV into a variant. A mammalian MPV can beclassified as an MPV variant A1, if the amino acid sequence of its Gprotein is at least 66%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, or at least 99% orat least 99.5% identical to the G protein of a mammalian MPV variant A1as represented by the prototype NL/1/00 (SEQ ID NO:322); if the aminoacid sequence of its N protein is at least 99.5% identical to the Nprotein of a mammalian MPV variant A1 as represented by the prototypeNL/1/00 (SEQ ID NO:366); if the amino acid sequence of its P protein isat least 96%, at least 98%, or at least 99% or at least 99.5% identicalto the P protein of a mammalian MPV variant A1 as represented by theprototype NL/1/00 (SEQ ID NO:374); if the amino acid sequence of its Mprotein is at least 99% or at least 99.5% identical to the M protein ofa mammalian MPV variant A1 as represented by the prototype NL/1/00 (SEQID NO:358); if the amino acid sequence of its F protein is at least 98%or at least 99% or at least 99.5% identical to the F protein of amammalian MPV variant A1 as represented by the prototype NL/1/00 (SEQ IDNO:314); if the amino acid sequence of its M2-1 protein is at least 99%or at least 99.5% identical to the M2-1 protein of a mammalian MPVvariant A1 as represented by the prototype NL/1/00 (SEQ ID NO:338); ifthe amino acid sequence of its M2-2 protein is at least 96% or at least99% or at least 99.5% identical to the M2-2 protein of a mammalian MPVvariant A1 as represented by the prototype NL/1/00 (SEQ ID NO:346); ifthe amino acid sequence of its SH protein is at least 84%, at least 90%,at least 95%, at least 98%, or at least 99% or at least 99.5% identicalto the SH protein of a mammalian MPV variant A1 as represented by theprototype NL/1/00 (SEQ ID NO:382); and/or if the amino acid sequence ofits L protein is at least 99% or at least 99.5% identical to the Lprotein of a virus of a mammalian MPV variant A1 as represented by theprototype NL/1/00 (SEQ ID NO:330).

An isolate of mammalian MPV is classified as a variant A2 if it isphylogenetically closer related to the viral isolate NL/17/00 (SEQ IDNO:20) than it is related to any of the following other viral isolates:NL/1/99 (SEQ ID NO:18), NL/1/00 (SEQ ID NO:19) and NL/1/94 (SEQ IDNO:21). One or more of the ORFs of a mammalian MPV can be used toclassify the mammalian MPV into a variant. A mammalian MPV can beclassified as an MPV variant A2, if the amino acid sequence of its Gprotein is at least 66%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99% or atleast 99.5% identical to the G protein of a mammalian MPV variant A2 asrepresented by the prototype NL/17/00 (SEQ ID NO:332); if the amino acidsequence of its N protein is at least 99.5% identical to the N proteinof a mammalian MPV variant A2 as represented by the prototype NL/17/00(SEQ ID NO:367); if the amino acid sequence of its P protein is at least96%, at least 98%, at least 99% or at least 99.5% identical to the Pprotein of a mammalian MPV variant A2 as represented by the prototypeNL/17/00 (SEQ ID NO:375); if the amino acid sequence of its M protein isat least 99%, or at least 99.5% identical to the M protein of amammalian MPV variant A2 as represented by the prototype NL/17/00 (SEQID NO:359); if the amino acid sequence of its F protein is at least 98%,at least 99% or at least 99.5% identical to the F protein of a mammalianMPV variant A2 as represented by the prototype NL/17/00 (SEQ ID NO:315);if the amino acid sequence of its M2-1 protein is at least 99%, or atleast 99.5% identical to the M2-1 protein of a mammalian MPV variant A2as represented by the prototype NL/17/00 (SEQ ID NO:339); if the aminoacid sequence of its M2-2 protein is at least 96%, at least 98%, atleast 99% or at least 99.5% identical to the M2-2 protein of a mammalianMPV variant A2 as represented by the prototype NL/17/00 (SEQ ID NO:347);if the amino acid sequence of its SH protein is at least 84%, at least85%, at least 90%, at least 95%, at least 98%, at least 99% or at least99.5% identical to the SH protein of a mammalian MPV variant A2 asrepresented by the prototype NL17/00 (SEQ ID NO:383); if the amino acidsequence of its L protein is at least 99% or at least 99.5% identical tothe L protein of a mammalian MPV variant A2 as represented by theprototype NL/17/00 (SEQ ID NO:331).

An isolate of mammalian MPV is classified as a variant B2 if it isphylogenetically closer related to the viral isolate NL/1/94 (SEQ IDNO:21) than it is related to any of the following other viral isolates:NL/1/99 (SEQ ID NO:18), NL/1/00 (SEQ ID NO:19) and NL/17/00 (SEQ IDNO:20). One or more of the ORFs of a mammalian MPV can be used toclassify the mammalian MPV into a variant. A mammalian MPV can beclassified as an MPV variant B2, if the amino acid sequence of its Gprotein is at least 66%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, or at least 99% orat least 99.5% identical to the G protein of a mammalian MPV variant B2as represented by the prototype NL/1/94 (SEQ ID NO:325); if the aminoacid sequence of its N protein is at least 99% or at least 99.5%identical to the N protein of a mammalian MPV variant B2 as representedby the prototype NL/1/94 (SEQ ID NO:369); if the amino acid sequence ofits P protein is at least 96%, at least 98%, or at least 99% or at least99.5% identical to the P protein of a mammalian MPV variant B2 asrepresented by the prototype NL/1/94 (SEQ ID NO:377); if the amino acidsequence of its M protein is identical to the M protein of a mammalianMPV variant B2 as represented by the prototype NL/1/94 (SEQ ID NO:361);if the amino acid sequence of its F protein is at least 99% or at least99.5% identical to the F protein of a mammalian MPV variant B2 asrepresented by the prototype NL/1/94 (SEQ ID NO:317); if the amino acidsequence of the M2-1 protein is at least 98% or at least 99% or at least99.5% identical to the M2-1 protein of a mammalian MPV variant B2 asrepresented by the prototype NL/1/94 (SEQ ID NO:341); if the amino acidsequence that is at least 99% or at least 99.5% identical to the M2-2protein of a mammalian MPV variant B2 as represented by the prototypeNL/1/94 (SEQ ID NO:349); if the amino acid sequence of its SH protein isat least 84%, at least 85%, at least 90%, at least 95%, at least 98%, orat least 99% or at least 99.5% identical to the SH protein of amammalian MPV variant B2 as represented by the prototype NL/1/94 (SEQ IDNO:385); and/or if the amino acid sequence of its L protein is at least99% or at least 99.5% identical to the L protein of a mammalian MPVvariant B2 as represented by the prototype NL/1/94 (SEQ ID NO:333).

In certain embodiments, the percentage of sequence identity is based onan alignment of the full length proteins. In other embodiments, thepercentage of sequence identity is based on an alignment of contiguousamino acid sequences of the proteins, wherein the amino acid sequencescan be 25 amino acids, 50 amino acids, 75 amino acids, 100 amino acids,125 amino acids, 150 amino acids, 175 amino acids, 200 amino acids, 225amino acids, 250 amino acids, 275 amino acids, 300 amino acids, 325amino acids, 350 amino acids, 375 amino acids, 400 amino acids, 425amino acids, 450 amino acids, 475 amino acids, 500 amino acids, 750amino acids, 1000 amino acids, 1250 amino acids, 1500 amino acids, 1750amino acids, 2000 amino acids or 2250 amino acids in length.

5.2 Functional Characteristics of a Mammalian MPV

In addition to the structural definitions of the mammalian MPV, amammalian MPV can also be defined by its functional characteristics. Incertain embodiments, the mammalian MPV of the invention is capable ofinfecting a mammalian host. The mammalian host can be a mammalian cell,tissue, organ or a mammal. In a specific embodiment, the mammalian hostis a human or a human cell, tissue or organ. Any method known to theskilled artisan can be used to test whether the mammalian host has beeninfected with the mammalian MPV. In certain embodiments, the virus istested for its ability to attach to a mammalian cell. In certain otherembodiments, the virus is tested for its ability to transfer its genomeinto the mammalian cell. In an illustrative embodiment, the genome ofthe virus is detectably labeled, e.g., radioactively labeled. The virusis then incubated with a mammalian cell for at least 1 minute, at least5 minutes at least 15 minutes, at least 30 minutes, at least 1 hour, atleast 2 hours, at least 5 hours, at least 12 hours, or at least 1 day.The cells are subsequently washed to remove any viral particles from thecells and the cells are then tested for the presence of the viral genomeby virtue of the detectable label. In another embodiment, the presenceof the viral genome in the cells is detected using RT-PCR usingmammalian MPV specific primers. (See, PCT WO 02/057302 at pp. 37 to 44,which is incorporated by reference herein.)

In certain embodiments, the mammalian virus is capable to infect amammalian host and to cause proteins of the mammalian MPV to be insertedinto the cytoplasmic membrane of the mammalian host. The mammalian hostcan be a cultured mammalian cell, organ, tissue or mammal. In anillustrative embodiment, a mammalian cell is incubated with themammalian virus. The cells are subsequently washed under conditions thatremove the virus from the surface of the cell. Any technique known tothe skilled artisan can be used to detect the newly expressed viralprotein inserted in the cytoplasmic membrane of the mammalian cell. Forexample, after infection of the cell with the virus, the cells aremaintained in medium comprising a detectably labeled amino acid. Thecells are subsequently harvested, lysed, and the cytoplasmic fraction isseparated from the membrane fraction. The proteins of the membranefraction are then solubilized and then subjected to animmunoprecipitation using antibodies specific to a protein of themammalian MPV, such as, but not limited to, the F protein or the Gprotein. The immunoprecipitated proteins are then subjected to SDS PAGE.The presence of viral protein can then be detected by autoradiography.In another embodiment, the presence of viral proteins in the cytoplasmicmembrane of the host cell can be detected by immunocytochemistry usingone or more antibodies specific to proteins of the mammalian MPV.

In even other embodiments, the mammalian MPV of the invention is capableof infecting a mammalian host and of replicating in the mammalian host.The mammalian host can be a cultured mammalian cell, organ, tissue ormammal. Any technique known to the skilled artisan can be used todetermine whether a virus is capable of infecting a mammalian cell andof replicating within the mammalian host. In a specific embodiment,mammalian cells are infected with the virus. The cells are subsequentlymaintained for at least 30 minutes, at least 1 hour, at least 2 hours,at least 5 hours, at least 12 hours, at least 1 day, or at least 2 days.The level of viral genomic RNA in the cells can be monitored usingNorthern blot analysis, RT-PCR or in situ hybridization using probesthat are specific to the viral genome. An increase in viral genomic RNAdemonstrates that the virus can infect a mammalian cell and canreplicate within a mammalian cell.

In even other embodiments, the mammalian MPV of the invention is capableof infecting a mammalian host, wherein the infection causes themammalian host to produce new infectious mammalian MPV. The mammalianhost can be a cultured mammalian cell or a mammal. Any technique knownto the skilled artisan can be used to determine whether a virus iscapable of infecting a mammalian host and cause the mammalian host toproduce new infectious viral particles. In an illustrative example,mammalian cells are infected with a mammalian virus. The cells aresubsequently washed and incubated for at least 30 minutes, at least 1hour, at least 2 hours, at least 5 hours, at least 12 hours, at least 1day, at least 2 days, at least one week, or at least twelve days. Thetiter of virus can be monitored by any method known to the skilledartisan. For exemplary methods see section 5.8.

In certain, specific embodiments, the mammalian MPV is a human MPV. Thetests described in this section can also be performed with a human MPV.In certain embodiments, the human MPV is capable of infecting amammalian host, such as a mammal or a mammalian cultured cell.

In certain embodiments, the human MPV is capable to infect a mammalianhost and to cause proteins of the human MPV to be inserted into thecytoplasmic membrane of the mammalian host.

In even other embodiments, the human MPV of the invention is capable ofinfecting a mammalian host and of replicating in the mammalian host.

In even other embodiments, the human MPV of the invention is capable ofinfecting a mammalian host and of replicating in the mammalian host,wherein the infection and replication causes the mammalian host toproduce and package new infectious human MPV.

In certain embodiments, the mammalian MPV, even though it is capable ofinfecting a mammalian host, is also capable of infecting an avian host,such as a bird or an avian cultured cell. In certain embodiments, themammalian MPV is capable to infect an avian host and to cause proteinsof the mammalian MPV to be inserted into the cytoplasmic membrane of theavian host. In even other embodiments, the mammalian MPV of theinvention is capable of infecting an avian host and of replicating inthe avian host. In even other embodiments, the mammalian MPV of theinvention is capable of infecting an avian host and of replicating inthe avian host, wherein the infection and replication causes the avianhost to produce and package new infectious mammalian MPV.

5.3 Recombinant and Chimeric Metapneumovirus

The present invention encompasses recombinant or chimeric virusesencoded by viral vectors derived from the genomes of metapneumovirus,including both mammalian and avian variants. In accordance with thepresent invention a recombinant virus is one derived from a mammalianMPV or an APV that is encoded by endogenous or native genomic sequencesor non-native genomic sequences. In accordance with the invention, anon-native sequence is one that is different from the native orendogenous genomic sequence due to one or more mutations, including, butnot limited to, point mutations, rearrangements, insertions, deletionsetc., to the genomic sequence that may or may not result in a phenotypicchange. The recombinant viruses of the invention encompass those virusesencoded by viral vectors derived from the genomes of metapneumovirus,including both mammalian and avian variants, and may or may not, includenucleic acids that are non-native to the viral genome. In accordancewith the present invention, a viral vector which is derived from thegenome of a metapneumovirus is one that contains a nucleic acid sequencethat encodes at least a part of one ORF of a mammalian metapneumovirus,wherein the polypeptides encoded by the ORF have amino acid sequenceidentity as set forth in Section 5.1 supra, and Table 1.

In accordance with the present invention, the recombinant viruses of theinvention encompass those viruses encoded by viral vectors derived fromthe genome of a mammalian metapneumovirus (MPV), in particular a humanmetapneumovirus. In particular embodiments of the invention, the viralvector is derived from the genome of a metapneumovirus A1, A2, B1 or B2variant. In accordance with the present invention, these viral vectorsmay or may not include nucleic acids that are non-native to the viralgenome

In accordance with the present invention, the recombinant viruses of theinvention encompass those viruses encoded by viral vectors derived fromthe genome of an avian pneumovirus (APV), also known as turkeyrhinotracheitis virus (TRTV). In particular embodiments of theinvention, the viral vector is derived from the genome of an APVsubgroup A, B, C or D. In a preferred embodiment, a viral vector derivedfrom the genome of an APV subgroup C. In accordance with the presentinvention these viral vectors may or may not include nucleic acids thatare non-native to the viral genome.

In another preferred embodiment of the invention, the recombinantviruses of the invention encompass those viruses encoded by a viralvector derived from the genome of an APV that contains a nucleic acidsequence that encodes a F-ORF of APV subgroup C. In certain embodiments,a viral vector derived from the genome of an APV is one that contains anucleic acid sequence that encodes at least a N-ORF, a P-ORF, a M-ORF, aF-ORF, a M2-1-ORF, a M2-2-ORF or a L-ORF of APV.

In accordance with the invention, a chimeric virus is a recombinant MPVor APV which further comprises a heterologous nucleotide sequence. Inaccordance with the invention, a chimeric virus may be encoded by anucleotide sequence in which heterologous nucleotide sequences have beenadded to the genome or in which endogenous or native nucleotidesequences have been replaced with heterologous nucleotide sequences.

In accordance with the invention, the chimeric viruses are encoded bythe viral vectors of the invention which further comprise a heterologousnucleotide sequence. In accordance with the present invention a chimericvirus is encoded by a viral vector that may or may not include nucleicacids that are non-native to the viral genome. In accordance with theinvention a chimeric virus is encoded by a viral vector to whichheterologous nucleotide sequences have been added, inserted orsubstituted for native or non-native sequences. In accordance with thepresent invention, the chimeric virus may be encoded by nucleotidesequences derived from different strains of mammalian MPV. Inparticular, the chimeric virus is encoded by nucleotide sequences thatencode antigenic polypeptides derived from different strains of MPV.

In accordance with the present invention, the chimeric virus may beencoded by a viral vector derived from the genome of an APV, inparticular subgroup C, that additionally encodes a heterologous sequencethat encodes antigenic polypeptides derived from one or more strains ofMPV.

A chimeric virus may be of particular use for the generation ofrecombinant vaccines protecting against two or more viruses (Tao et al.,J. Virol. 72, 2955-2961; Durbin et al., 2000, J. Virol. 74, 6821-6831;Skiadopoulos et al., 1998, J. Virol. 72, 1762-1768; Teng et al., 2000,J. Virol. 74, 9317-9321). For example, it can be envisaged that a MPV orAPV virus vector expressing one or more proteins of another negativestrand RNA virus, e.g., RSV or a RSV vector expressing one or moreproteins of MPV will protect individuals vaccinated with such vectoragainst both virus infections. A similar approach can be envisaged forPly or other paramyxoviruses. Attenuated and replication-defectiveviruses may be of use for vaccination purposes with live vaccines as hasbeen suggested for other viruses. (See, PCT WO 02/057302, at pp. 6 and23, incorporated by reference herein.)

In accordance with the present invention the heterologous sequence to beincorporated into the viral vectors encoding the recombinant or chimericviruses of the invention include sequences obtained or derived fromdifferent strains of metapneumovirus, strains of avian pneumovirus, andother negative strand RNA viruses, including, but not limited to, RSV,PIV and influenza virus, and other viruses, including morbillivirus.

In certain embodiments of the invention, the chimeric or recombinantviruses of the invention are encoded by viral vectors derived from viralgenomes wherein one or more sequences, intergenic regions, terminisequences, or portions or entire ORF have been substituted with aheterologous or non-native sequence. In certain embodiments of theinvention, the chimeric viruses of the invention are encoded by viralvectors derived from viral genomes wherein one or more heterologoussequences have been added to the vector.

In certain embodiments, the virus of the invention contains heterologousnucleic acids. In a preferred embodiment, the heterologous nucleotidesequence is inserted or added at Position 1 of the viral genome. Inanother preferred embodiment, the heterologous nucleotide sequence isinserted or added at Position 2 of the viral genome. In even anotherpreferred embodiment, the heterologous nucleotide sequence is insertedor added at Position 3 of the viral genome. Insertion or addition ofnucleic acid sequences at the lower-numbered positions of the viralgenome results in stronger or higher levels of expression of theheterologous nucleotide sequence compared to insertion athigher-numbered positions due to a transcriptional gradient across thegenome of the virus. Thus, inserting or adding heterologous nucleotidesequences at lower-numbered positions is the preferred embodiment of theinvention if high levels of expression of the heterologous nucleotidesequence is desired.

Without being bound by theory, the position of insertion or addition ofthe heterologous sequence affects the replication rate of therecombinant or chimeric virus. The higher rates of replication can beachieved if the heterologous sequence is inserted or added at Position 2or Position 1 of the viral genome. The rate of replication is reduced ifthe heterologous sequence is inserted or added at Position 3, Position4, Position 5, or Position 6.

Without being bound by theory, the size of the intergenic region betweenthe viral gene and the heterologous sequence further determines rate ofreplication of the virus and expression levels of the heterologoussequence.

In certain embodiments, the viral vector of the invention contains twoor more different heterologous nucleotide sequences. In a preferredembodiment, one heterologous nucleotide sequence is at Position 1 and asecond heterologous nucleotide sequence is a Position 2 of the viralgenome. In another preferred embodiment, one heterologous nucleotidesequence is at Position 1 and a second heterologous nucleotide sequenceis at Position 3 of the viral genome. In even another preferredembodiment, one homologous nucleotide sequence is at Position 2 and asecond heterologous nucleotide sequence is at Position 3 of the viralgenome. In certain other embodiments, a heterologous nucleotide sequenceis inserted at other, higher-numbered positions of the viral genome. Inaccordance with the present invention, the position of the heterologoussequence refers to the order in which the sequences are transcribed fromthe viral genome, e.g., a heterologous sequence at Position 1 is thefirst gene sequence to be transcribed from the genome.

The selection of the viral vector may depend on the species of thesubject that is to be treated or protected from a viral infection. Ifthe subject is human, then an attenuated mammalian metapneumovirus or anavian pneumovirus can be used to provide the antigenic sequences.

In accordance with the present invention, the viral vectors can beengineered to provide antigenic sequences which confer protectionagainst infection by a metapneumovirus, including sequences derived frommammalian metapneumovirus human metapneumovirus, MPV variants A1, A2, B1or B2, sequences derived from avian pneumovirus including APV subgroupsA, B, C or D, although C is preferred. The viral vectors can beengineered to provide antigenic sequences which confer protectionagainst infection or disease by another virus, including negative strandRNA virus, including influenza, RSV or PW, including PIV3. The viralvectors may be engineered to provide one, two, three or more antigenicsequences. In accordance with the present invention the antigenicsequences may be derived from the same virus, from different strains orvariants of the same type of virus, or from different viruses, includingmorbillivirus.

In certain embodiments of the invention, the heterologous nucleotidesequence to be inserted into the genome of the virus of the invention isderived from a metapneumovirus. In certain specific embodiments of theinvention, the heterologous nucleotide sequence is derived from a humanmetapneumovirus. In another specific embodiment, the heterologousnucleotide sequence is derived from an avian pneumovirus. Morespecifically, the heterologous nucleotide sequence of the inventionencodes a F gene of a human metapneumovirus. More specifically, theheterologous nucleotide sequence of the invention encodes an G gene of ahuman metapneumovirus. More specifically, the heterologous nucleotidesequence of the invention encodes a F gene of an avian pneumovirus. Morespecifically, the heterologous nucleotide sequence of the inventionencodes a G gene of an avian pneumovirus. In specific embodiments, aheterologous nucleotide sequences can be any one of SEQ ID NO:1 throughSEQ ID NO:5, SEQ ID NO:14, and SEQ ID NO:15. In certain specificembodiments, the nucleotide sequence encodes a protein of any one of SEQID NO:6 through SEQ ID NO:13, SEQ ID NO:16, and SEQ ID NO:17.

In a specific embodiment of the invention, the heterologous nucleotidesequence encodes a chimeric F protein. In an illustrative embodiment,the ectodomain of the chimeric F-protein is the ectodomain of a humanMPV and the transmembrane domain and the luminal domain are derived fromthe F-protein of an avian metapneumovirus. Without being bound bytheory, a chimeric human MPV that encodes the chimeric F-proteinconsisting of the human ectodomain and the avian luminol/transmembranedomain is attenuated because of the avian part of the F-protein, yethighly immunogenic against hMPV because of the human ectodomain.

In certain embodiments, two different heterologous nucleotide sequencesare inserted or added to the viral vectors of the invention, derivedfrom metapneumoviral genomes, including mammalian and avian. Forexample, the heterologous nucleotide sequence is derived from a humanmetapneumovirus, an avian pneumovirus, RSV, PIV, or influenza. In apreferred embodiment, the heterologous sequence encodes the F-protein ofhuman metapneumovirus, avian pneumovirus, RSV or PIV respectively. Inanother embodiment, the heterologous sequence encodes the HA protein ofinfluenza.

In certain embodiments, the viral vector of the invention contains twodifferent heterologous nucleotide sequences wherein a first heterologousnucleotide sequence is derived from a metapneumovirus, such as a humanmetapneumovirus or an avian pneumovirus, and a second nucleotidesequence is derived from a respiratory syncytial virus (see Table 2). Inspecific embodiments, the heterologous nucleotide sequence derived fromrespiratory syncytial virus is a F gene of a respiratory syncytialvirus. In other specific embodiments, the heterologous nucleotidesequence derived from respiratory syncytial virus is a G gene of arespiratory syncytial virus. In a specific embodiment, the heterologousnucleotide sequence derived from a metapneumovirus is inserted at alower-numbered position than the heterologous nucleotide sequencederived from a respiratory syncytial virus.

In another specific embodiment, the heterologous nucleotide sequencederived from a metapneumovirus is inserted at a higher-numbered positionthan the heterologous nucleotide sequence derived from a respiratorysyncytial virus.

In certain embodiments, the virus of the invention contains twodifferent heterologous nucleotide sequences wherein a first heterologousnucleotide sequence is derived from a metapneumovirus, such as a humanmetapneumovirus or an avian pneumovirus, and a second nucleotidesequence is derived from a parainfluenza virus, such as, but not limitedto PIV3 (see Table 2). In specific embodiments, the heterologousnucleotide sequence derived from PIV is a F gene of Ply. In otherspecific embodiments, the heterologous nucleotide sequence derived fromPIV is a G gene of a PIV. In a specific embodiment, the heterologousnucleotide sequence derived from a metapneumovirus is inserted at alower-numbered position than the heterologous nucleotide sequencederived from a PIV. In another specific embodiment, the heterologousnucleotide sequence derived from a metapneumovirus is inserted at ahigher-numbered position than the heterologous nucleotide sequencederived from a PIV.

The expression products and/or recombinant or chimeric virions obtainedin accordance with the invention may advantageously be utilized invaccine formulations. The expression products and chimeric virions ofthe present invention may be engineered to create vaccines against abroad range of pathogens, including viral and bacterial antigens, tumorantigens, allergen antigens, and auto antigens involved in autoimmunedisorders. In particular, the chimeric virions of the present inventionmay be engineered to create vaccines for the protection of a subjectfrom infections with PIV, RSV, and/or metapneumovirus.

In another embodiment, the chimeric virions of the present invention maybe engineered to create anti-HIV vaccines, wherein an immunogenicpolypeptide from gp 160, and/or from internal proteins of HIV isengineered into the glycoprotein HN protein to construct a vaccine thatis able to elicit both vertebrate humoral and cell-mediated immuneresponses. In yet another embodiment, the invention relates torecombinant metapneumoviral vectors and viruses which are engineered toencode mutant antigens. A mutant antigen has at least one amino acidsubstitution, deletion or addition relative to the wild-type viralprotein from which it is derived.

In certain embodiments, the invention relates to trivalent vaccinescomprising a recombinant or chimeric virus of the invention. In specificembodiments, the virus used as backbone for a trivalent vaccine is achimeric avian-human metapneumovirus or a chimeric human-avianmetapneumovirus containing a first heterologous nucleotide sequencederived from a RSV and a second heterologous nucleotide sequence derivedfrom PIV. In an exemplary embodiment, such a trivalent vaccine will bespecific to (a) the gene products of the F gene and/or the G gene of thehuman metapneumovirus or avian pneumovirus, respectively, dependent onwhether chimeric avian-human or chimeric human-avian metapneumovirus isused; (b) the protein encoded by the heterologous nucleotide sequencederived from a RSV; and (c) the protein encoded by the heterologousnucleotide sequence derived from Ply. In a specific embodiment, thefirst heterologous nucleotide sequence is the F gene of the respiratorysyncytial virus and is inserted in Position 1, and the secondheterologous nucleotide sequence is the F gene of the PIV and isinserted in Position 3. Many more combinations are encompassed by thepresent invention and some are shown by way of example in Table 2.Further, nucleotide sequences encoding chimeric F proteins could be used(see supra). In some less preferred embodiments, the heterologousnucleotide sequence can be inserted at higher-numbered positions of theviral genome.

TABLE 2 Exemplary arrangements of heterologous nucleotide sequences inthe viruses used for trivalent vaccines. Combination Position 1 Position2 Position 3 1 F-gene of PIV F-gene of RSV — 2 F-gene of RSV F-gene ofPIV — 3 — F-gene of PIV F-gene of RSV 4 — F-gene of RSV F-gene of PIV 5F-gene of PIV — F-gene of RSV 6 F-gene of RSV — F-gene of PIV 7 HN-geneof PIV G-gene of RSV — 8 G-gene of RSV HN-gene of PIV — 9 — HN-gene ofPIV G-gene of RSV 10 — G-gene of RSV HN-gene of PIV 11 HN-gene of PIV —G-gene of RSV 12 G-gene of RSV — HN-gene of PIV 13 F-gene of PIV G-geneof RSV — 14 G-gene of RSV F-gene of PIV — 15 — F-gene of PIV G-gene ofRSV 16 — G-gene of RSV F-gene of PIV 17 F-gene of PIV — G-gene of RSV 18G-gene of RSV — F-gene of PIV 19 HN-gene of PIV F-gene of RSV — 20F-gene of RSV HN-gene of PIV — 21 — HN-gene of PIV F-gene of RSV 22 —F-gene of RSV HN-gene of PIV 23 HN-gene of PIV — F-gene of RSV 24 F-geneof RSV — HN-gene of PIV

In certain embodiments, the expression products and recombinant orchimeric virions of the present invention may be engineered to createvaccines against a broad range of pathogens, including viral antigens,tumor antigens and auto antigens involved in autoimmune disorders. Oneway to achieve this goal involves modifying existing metapneumoviralgenes to contain foreign sequences in their respective external domains.Where the heterologous sequences are epitopes or antigens of pathogens,these chimeric viruses may be used to induce a protective immuneresponse against the disease agent from which these determinants arederived.

Thus, the present invention relates to the use of viral vectors andrecombinant or chimeric viruses to formulate vaccines against a broadrange of viruses and/or antigens. The viral vectors and chimeric virusesof the present invention may be used to modulate a subject's immunesystem by stimulating a humoral immune response, a cellular immuneresponse or by stimulating tolerance to an antigen. As used herein, asubject means: humans, primates, horses, cows, sheep, pigs, goats, dogs,cats, avian species and rodents.

The invention may be divided into the following stages solely for thepurpose of description and not by way of limitation: (a) construction ofrecombinant cDNA and RNA templates; (b) expression of heterologous geneproducts using recombinant cDNA and RNA templates; (c) rescue of theheterologous gene in recombinant virus particles; and (d) generation anduse of vaccines comprising the recombinant virus particles of theinvention.

5.4 Construction of the Recombinant cDNA and RNA

In certain embodiments, the viral vectors are derived from the genomesof human or mammalian metapneumovirus of the invention. In otherembodiments, the viral vectors are derived from the genome of avianpneumovirus. In certain embodiments, viral vectors contain sequencesderived from mammalian MPV and APV, such that a chimeric human MPV/APVvirus is encoded by the viral vector. In an exemplary embodiment, theF-gene and/or the G-gene of human metapneumovirus have been replacedwith the F-gene and/or the G-gene of avian pneumovirus to constructchimeric hMPV/APV virus. In other embodiments, viral vectors containsequences derived from APV and mammalian MPV, such that a chimericAPV/hMPV virus is encoded by the viral vector. In more exemplaryembodiments, the F-gene and/or the G-gene of avian pneumovirus have beenreplaced with the F-gene and/or the G-gene of human metapneumovirus toconstruct the chimeric APV/hMPV virus.

The present invention also encompasses recombinant viruses comprising aviral vector derived from a mammalian MPV or APV genome containingsequences endogenous or native to the viral genome, and may or may notcontain sequences non-native to the viral genome. Non-native sequencesinclude those that are different from native or endogenous sequenceswhich may or may not result in a phenotypic change. The recombinantviruses of the invention may contain sequences which result in a virushaving a phenotype more suitable for use in vaccine formulations, e.g.,attenuated phenotype or enhanced antigenicity. The mutations andmodifications can be in coding regions, in intergenic regions and in theleader and trailer sequences of the virus.

In certain embodiments the viral vectors of the invention comprisenucleotide sequences derived from hMPV, APV, hMPV/APV or APV/hMPV, inwhich native nucleotide sequences have been substituted withheterologous sequences or in which heterologous sequences have beenadded to the native metapneumoviral sequences.

In a more specific embodiment, a chimeric virus comprises a viral vectorderived from MPV, APV, APV/hMPV, or hMPV/APV in which heterologoussequences derived from PIV have been added. In a more specificembodiment, a recombinant virus comprises a viral vector derived fromMPV, APV, APV/hMPV, or hMPV/APV in which sequences have been replaced byheterologous sequences derived from PIV. In other specific embodiments,a chimeric virus comprises a viral vector derived from MPV, APV,APV/hMPV, or hMPV/APV in which heterologous sequences derived from RSVhave been added. In a more specific embodiment, a chimeric viruscomprises a viral vector derived from MPV, APV, APV/hMPV, or hMPV/APV inwhich sequences have been replaced by heterologous sequences derivedfrom RSV.

Heterologous gene coding sequences flanked by the complement of theviral polymerase binding site/promoter, e.g., the complement of 3′-hMPVvirus terminus of the present invention, or the complements of both the3′- and 5′-hMPV virus termini may be constructed using techniques knownin the art. In more specific embodiments, a recombinant virus of theinvention contains the leader and trailer sequence of hMPV or APV. Incertain embodiments, the intergenic regions are obtained from hMPV orAPV. The resulting RNA templates may be of the negative-polarity andcontain appropriate terminal sequences which enable the viralRNA-synthesizing apparatus to recognize the template. Alternatively,positive-polarity RNA templates which contain appropriate terminalsequences which enable the viral RNA-synthesizing apparatus to recognizethe template, may also be used. Recombinant DNA molecules containingthese hybrid sequences can be cloned and transcribed by a DNA-directedRNA polymerase, such as bacteriophage T7, T3, the SP6 polymerase oreukaryotic polymerase such as polymerase I and the like, to produce invitro or in vivo the recombinant RNA templates which possess theappropriate viral sequences that allow for viral polymerase recognitionand activity. In a more specific embodiment, the RNA polymerase isfowlpox virus T7 RNA polymerase or a MVA T7 RNA polymerase.

An illustrative approach for constructing these hybrid molecules is toinsert the heterologous nucleotide sequence into a DNA complement of ahMPV, APV, APV/hMPV or hMPV/APV genome, so that the heterologoussequence is flanked by the viral sequences required for viral polymeraseactivity, i.e., the viral polymerase binding site/promoter, hereinafterreferred to as the viral polymerase binding site, and a polyadenylationsite. In a preferred embodiment, the heterologous coding sequence isflanked by the viral sequences that comprise the replication promotersof the 5′ and 3′ termini, the gene start and gene end sequences, and thepackaging signals that are found in the 5′ and/or the 3′ termini. In analternative approach, oligonucleotides encoding the viral polymerasebinding site, e.g., the complement of the 3′-terminus or both termini ofthe virus genomic segment can be ligated to the heterologous codingsequence to construct the hybrid molecule. The placement of a foreigngene or segment of a foreign gene within a target sequence was formerlydictated by the presence of appropriate restriction enzyme sites withinthe target sequence. However, recent advances in molecular biology havelessened this problem greatly. Restriction enzyme sites can readily beplaced anywhere within a target sequence through the use ofsite-directed mutagenesis (e.g., see, for example, the techniquesdescribed by Kunkel, 1985, Proc. Natl. Acad. Sci. U.S.A. 82; 488).Variations in polymerase chain reaction (PCR) technology, describedinfra, also allow for the specific insertion of sequences (i.e.,restriction enzyme sites) and allow for the facile construction ofhybrid molecules. Alternatively, PCR reactions could be used to preparerecombinant templates without the need of cloning. For example, PCRreactions could be used to prepare double-stranded DNA moleculescontaining a DNA-directed RNA polymerase promoter (e.g., bacteriophageT3, T7 or SP6) and the hybrid sequence containing the heterologous geneand the PIV polymerase binding site. RNA templates could then betranscribed directly from this recombinant DNA. In yet anotherembodiment, the recombinant RNA templates may be prepared by ligatingRNAs specifying the negative polarity of the heterologous gene and theviral polymerase binding site using an RNA ligase.

In addition, one or more nucleotides can be added in the untranslatedregion to adhere to the “Rule of Six” which may be important inobtaining virus rescue. The “Rule of Six” applies to manyparamyxoviruses and states that the RNA nucleotide genome must bedivisible by six to be functional. The addition of nucleotides can beaccomplished by techniques known in the art such as using a commercialmutagenesis kits such as the QuikChange mutagenesis kit (Stratagene).After addition of the appropriate number of nucleotides, the correct DNAfragment can then be isolated by digestion with appropriate restrictionenzyme and gel purification. Sequence requirements for viral polymeraseactivity and constructs which may be used in accordance with theinvention are described in the subsections below.

Without being bound by theory, several parameters affect the rate ofreplication of the recombinant virus and the level of expression of theheterologous sequence. In particular, the position of the heterologoussequence in hMPV, APV, hMPV/APV or APV/hMPV and the length of theintergenic region that flanks the heterologous sequence determine rateof replication and expression level of the heterologous sequence.

In certain embodiments, the leader and or trailer sequence of the virusare modified relative to the wild type virus. In certain more specificembodiments, the lengths of the leader and/or trailer are altered. Inother embodiments, the sequence(s) of the leader and/or trailer aremutated relative to the wild type virus. For more detail, see section5.7.

The production of a recombinant virus of the invention relies on thereplication of a partial or full-length copy of the negative sense viralRNA (vRNA) genome or a complementary copy thereof (cRNA). This vRNA orcRNA can be isolated from infectious virus, produced upon in vitrotranscription, or produced in cells upon transfection of nucleic acids.Second, the production of recombinant negative strand virus relies on afunctional polymerase complex. Typically, the polymerase complex ofpneumoviruses consists of N, P, L and possibly M2 proteins, but is notnecessarily limited thereto.

Polymerase complexes or components thereof can be isolated from virusparticles, isolated from cells expressing one or more of the components,or produced upon transfection of specific expression vectors.

Infectious copies of MPV can be obtained when the above mentioned vRNA,cRNA, or vectors expressing these RNAs are replicated by the abovementioned polymerase complex 16 (Schnell et al., 1994, EMBO J. 13:4195-4203; Collins, et al., 1995, PNAS 92: 11563-11567; Hoffmann, etal., 2000, PNAS 97: 6108-6113; Bridgen, et al., 1996, PNAS 93:15400-15404; Palese, et al., 1996, PNAS 93: 11354-11358; Peeters, etal., 1999, J. Virol. 73: 5001-5009; Durbin, et al., 1997, Virology 235:323-332).

The invention provides a host cell comprising a nucleic acid or a vectoraccording to the invention. Plasmid or viral vectors containing thepolymerase components of MPV (presumably N, P, L and M2, but notnecessarily limited thereto) are generated in prokaryotic cells for theexpression of the components in relevant cell types (bacteria, insectcells, eukaryotic cells). Plasmid or viral vectors containingfull-length or partial copies of the MPV genome will be generated inprokaryotic cells for the expression of viral nucleic acids in vitro orin vivo. The latter vectors may contain other viral sequences for thegeneration of chimeric viruses or chimeric virus proteins, may lackparts of the viral genome for the generation of replication defectivevirus, and may contain mutations, deletions or insertions for thegeneration of attenuated viruses.

Infectious copies of MPV (being wild type, attenuated,replication-defective or chimeric) can be produced upon co-expression ofthe polymerase components according to the state-of-the-art technologiesdescribed above.

In addition, eukaryotic cells, transiently or stably expressing one ormore full-length or partial MPV proteins can be used. Such cells can bemade by transfection (proteins or nucleic acid vectors), infection(viral vectors) or transduction (viral vectors) and may be useful forcomplementation of mentioned wild type, attenuated,replication-defective or chimeric viruses.

5.4.1 Heterologous Gene Sequences to be Inserted

In accordance with the present invention the viral vectors of theinvention may be further engineered to express a heterologous sequence.In an embodiment of the invention, the heterologous sequence is derivedfrom a source other than the viral vector. By way of example, and not bylimitation, the heterologous sequence encodes an antigenic protein,polypeptide or peptide of a virus belonging to a different species,subgroup or variant of metapneumovirus than the species, subgroup orvariant from which the viral vector is derived. By way of example, andnot by limitation, the heterologous sequence encodes an antigenicprotein, polypeptide or peptide of a virus other than a metapneumovirus.By way of example, and not by limitation, the heterologous sequence isnot viral in origin. In accordance with this embodiment, theheterologous sequence may encode a moiety, peptide, polypeptide orprotein possessing a desired biological property or activity. Such aheterologous sequence may encode a tag or marker. Such a heterologoussequence may encode a biological response modifier, examples of whichinclude, lymphokines, interleukins, granulocyte macrophage colonystimulating factor and granulocyte colony stimulating factor.

In certain embodiments, the heterologous nucleotide sequence to beinserted is derived from a metapneumovirus. More specifically, theheterologous nucleotide sequence to be inserted is derived from a humanmetapneumovirus and/or an avian pneumovirus.

In certain embodiments, the heterologous sequence encodes PIVnucleocapsid phosphoprotein, PIV L protein, PIV matrix protein, PIV HNglycoprotein, PIV RNA-dependent RNA polymerase, PIV Y1 protein, PIV Dprotein, PIV C protein, PIV F protein or PIV P protein. In certainembodiments, the heterologous nucleotide sequence encodes a protein thatis at least 90%, at least 95%, at least 98%, or at least 99% homologousto PIV nucleocapsid phosphoprotein, PIV L protein, PIV matrix protein,PIV HN glycoprotein, PIV RNA-dependent RNA polymerase, PIV Y1 protein,PIV D protein, PIV C protein, PIV F protein or PIV P protein. Theheterologous sequence can be obtained from PIV type 1, PIV type 2, orPIV type 3. In more specific embodiments, the heterologous sequence isobtained from human PIV type 1, PTV type 2, or PIV type 3. In otherembodiments, the heterologous sequence encodes RSV nucleoprotein, RSVphosphoprotein, RSV matrix protein, RSV small hydrophobic protein, RSVRNA-dependent RNA polymerase, RSV F protein, RSV G protein, or RSV M2-1or M2-2 protein. In certain embodiments, the heterologous sequenceencodes a protein that is at least 90%, at least 95%, at least 98%, orat least 99% homologous to RSV nucleoprotein, RSV phosphoprotein, RSVmatrix protein, RSV small hydrophobic protein, RSV RNA-dependent RNApolymerase, RSV F protein, or RSV G protein. The heterologous sequencecan be obtained from RSV subtype A and RSV subtype B. In more specificembodiments, the heterologous sequence is obtained from human RSVsubtype A and RSV subtype B. In other embodiments, the heterologoussequence encodes APV nucleoprotein, APV phosphoprotein, APV matrixprotein, APV small hydrophobic protein, APV RNA-dependent RNApolymerase, APV F protein, APV G protein or APV M2-1 or M2-2 protein. Incertain embodiments, the heterologous sequence encodes a protein that isat least 90%, at least 95%, at least 98%, or at least 99% homologous toAPV nucleoprotein, APV phosphoprotein, APV matrix protein, APV smallhydrophobic protein, APV RNA-dependent RNA polymerase, APV F protein, orAPV G protein. The avian pneumovirus can be APV subgroup A, APV subgroupB, or APV subgroup C. In other embodiments, the heterologous sequenceencodes hMPV nucleoprotein, hMPV phosphoprotein, hMPV matrix protein,hMPV small hydrophobic protein, hMPV RNA-dependent RNA polymerase, hMPVF protein, hMPV G protein or hMPV M2-1 or M2-2. In certain embodiments,the heterologous sequence encodes a protein that is at least 90%, atleast 95%, at least 98%, or at least 99% homologous to hMPVnucleoprotein, hMPV phosphoprotein, hMPV matrix protein, hMPV smallhydrophobic protein, hMPV RNA-dependent RNA polymerase, hMPV F protein,or hMPV G protein. The human metapneumovirus can be hMPV variant A1,hMPV variant A2, hMPV variant B1, or hMPV variant B2.

In certain embodiments, any combination of different heterologoussequence from PIV, RSV, human metapneumovirus, or avian pneumovirus canbe inserted into the virus of the invention.

In certain preferred embodiments of the invention, the heterologousnucleotide sequence to be inserted is derived from a F gene from RSV,PIV, APV or hMPV.

In certain embodiments, the heterologous nucleotide sequence encodes achimeric protein. In more specific embodiments, the heterologousnucleotide sequence encodes a chimeric F protein of RSV, PIV, APV orhMPV. A chimeric F protein can comprise parts of F proteins fromdifferent viruses, such as a human metapneumovirus, avian pneumovirus,respiratory syncytial virus, and parainfluenza virus. In certain otherembodiments, the heterologous sequence encodes a chimeric G protein. Achimeric G protein comprises parts of G proteins from different viruses,such as a human metapneumovirus, avian pneumovirus, respiratorysyncytial virus, and parainfluenza virus. In a specific embodiment, theF protein comprises an ectodomain of a F protein of a metapneumovirus, atransmembrane domain of a F protein of a parainfluenza virus, andluminal domain of a F protein of a parainfluenza virus.

In certain specific embodiments, the heterologous nucleotide sequence ofthe invention is any one of SEQ ID NO:1 through SEQ ID NO:5, SEQ IDNO:14, and SEQ ID NO:15. In certain specific embodiments, the nucleotidesequence encodes a protein of any one of SEQ ID NO:6 through SEQ IDNO:13, SEQ ID NO:16, and SEQ ID NO:17.

For heterologous nucleotide sequences derived from respiratory syncytialvirus see, e.g., PCT/US98/20230, which is hereby incorporated byreference in its entirety.

In a preferred embodiment, heterologous gene sequences that can beexpressed into the recombinant viruses of the invention include but arenot limited to antigenic epitopes and glycoproteins of viruses whichresult in respiratory disease, such as influenza glycoproteins, inparticular hemagglutinin H5, H7, respiratory syncytial virus epitopes,New Castle Disease virus epitopes, Sendai virus and infectiousLaryngotracheitis virus (ILV). In a preferred embodiment, theheterologous nucleotide sequences are derived from a RSV or PIV. In yetanother embodiment of the invention, heterologous gene sequences thatcan be engineered into the chimeric viruses of the invention include,but are not limited to, viral epitopes and glycoproteins of viruses,such as hepatitis B virus surface antigen, hepatitis A or C virussurface glycoproteins of Epstein Barr virus, glycoproteins of humanpapilloma virus, simian virus 5 or mumps virus, West Nile virus, Denguevirus, glycoproteins of herpes viruses, VPI of poliovirus, and sequencesderived from a lentivirus, preferably, but not limited to humanimmunodeficiency virus (HIV) type 1 or type 2. In yet anotherembodiment, heterologous gene sequences that can be engineered intochimeric viruses of the invention include, but are not limited to,Marek's Disease virus (MDV) epitopes, epitopes of infectious BursalDisease virus (IBDV), epitopes of Chicken Anemia virus, infectiouslaryngotracheitis virus (ILV), Avian Influenza virus (AIV), rabies,feline leukemia virus, canine distemper virus, vesicular stomatitisvirus, and swinepox virus (see Fields et al., (ed.), 1991, FundamentalVirology, Second Edition, Raven Press, New York, incorporated byreference herein in its entirety).

Other heterologous sequences of the present invention include antigensthat are characteristic of autoimmune disease. These antigens willtypically be derived from the cell surface, cytoplasm, nucleus,mitochondria and the like of mammalian tissues, including antigenscharacteristic of diabetes mellitus, multiple sclerosis, systemic lupuserythematosus, rheumatoid arthritis, pernicious anemia, Addison'sdisease, scleroderma, autoimmune atrophic gastritis, juvenile diabetes,and discoid lupus erythromatosus.

Antigens that are allergens generally include proteins or glycoproteins,including antigens derived from pollens, dust, molds, spores, dander,insects and foods. In addition, antigens that are characteristic oftumor antigens typically will be derived from the cell surface,cytoplasm, nucleus, organelles and the like of cells of tumor tissue.Examples include antigens characteristic of tumor proteins, includingproteins encoded by mutated oncogenes; viral proteins associated withtumors; and glycoproteins. Tumors include, but are not limited to, thosederived from the types of cancer: lip, nasopharynx, pharynx and oralcavity, esophagus, stomach, colon, rectum, liver, gall bladder,pancreas, larynx, lung and bronchus, melanoma of skin, breast, cervix,uterine, ovary, bladder, kidney, uterus, brain and other parts of thenervous system, thyroid, prostate, testes, Hodgkin's disease,non-Hodgkin's lymphoma, multiple myeloma and leukemia.

In one specific embodiment of the invention, the heterologous sequencesare derived from the genome of human immunodeficiency virus (HIV),preferably human immunodeficiency virus-1 or human immunodeficiencyvirus-2. In another embodiment of the invention, the heterologous codingsequences may be inserted within a gene coding sequence of the viralbackbone such that a chimeric gene product is expressed which containsthe heterologous peptide sequence within the metapneumoviral protein. Insuch an embodiment of the invention, the heterologous sequences may alsobe derived from the genome of a human immunodeficiency virus, preferablyof human immunodeficiency virus-1 or human immunodeficiency virus-2.

In instances whereby the heterologous sequences are HIV-derived, suchsequences may include, but are not limited to sequences derived from theenv gene (i.e., sequences encoding all or part of gp160, gp120, and/orgp41), the pol gene (i.e., sequences encoding all or part of reversetranscriptase, endonuclease, protease, and/or integrase), the gag gene(i.e., sequences encoding all or part of p7, p6, p55, p17/18, p24/25)tat, rev, nef, vif, vpu, vpr, and/or vpx.

In yet another embodiment, heterologous gene sequences that can beengineered into the chimeric viruses include those that encode proteinswith immunopotentiating activities. Examples of immunopotentiatingproteins include, but are not limited to, cytokines, interferon type 1,gamma interferon, colony stimulating factors, and interleukin-1, -2, -4,-5, -6, -12.

In addition, other heterologous gene sequences that may be engineeredinto the chimeric viruses include antigens derived from bacteria such asbacterial surface glycoproteins, antigens derived from fungi, andantigens derived from a variety of other pathogens and parasites.Examples of heterologous gene sequences derived from bacterial pathogensinclude, but are not limited to, antigens derived from species of thefollowing genera: Salmonella, Shigella, Chlamydia, Helicobacter,Yersinia, Bordatella, Pseudomonas, Neisseria, Vibrio, Haemophilus,Mycoplasma, Streptomyces, Treponema, Coxiella, Ehrlichia, Brucella,Streptobacillus, Fusospirocheta, Spirillum, Ureaplasma, Spirochaeta,Mycoplasma, Actinomycetes, Borrelia, Bacteroides, Trichomoras,Branhamella, Pasteurella, Clostridium, Corynebacterium, Listeria,Bacillus, Erysipelothrix, Rhodococcus, Escherichia, Klebsiella,Pseudomanas, Enterobacter, Serratia, Staphylococcus, Streptococcus,Legionella, Mycobacterium, Proteus, Campylobacter, Enterococcus,Acinetobacter, Morganella, Moraxella, Citrobacter, Rickettsia,Rochlimeae, as well as bacterial species such as: P. aeruginosa; E.coli, P. cepacia, S. epidermis, E. faecalis, S. pneumonias, S. aureus, Nmeningitidis, S. pyogenes, Pasteurella multocida, Treponema pallidum,and P. mirabilis.

Examples of heterologous gene sequences derived from pathogenic fungi,include, but are not limited to, antigens derived from fungi such asCryptococcus neoformans; Blastomyces dermatitidis; Aiellomycesdermatitidis; Histoplasma capsulatum; Coccidioides immitis; Candidaspecies, including C. albicans, C. tropicalis, C. parapsilosis, C.guilliermondii and C. krusei, Aspergillus species, including A.fumigatus, A. flavus and A. niger, Rhizopus species; Rhizomucor species;Cunninghammella species; Apophysomyces species, including A. saksenaea,A. mucor and A. absidia; Sporothrix schenckii, Paracoccidioidesbrasiliensis; Pseudallescheria boydii, Torulopsis glabrata; Trichophytonspecies, Microsporum species and Dermatophyres species, as well as anyother yeast or fungus now known or later identified to be pathogenic.

Finally, examples of heterologous gene sequences derived from parasitesinclude, but are not limited to, antigens derived from members of theApicomplexa phylum such as, for example, Babesia, Toxoplasma,Plasmodium, Eimeria, Isospora, Atoxoplasma, Cystoisospora, Hammondia,Besniotia, Sarcocystis, Frenkelia, Haemoproteus, Leucocytozoon,Theileria, Perkinsus and Gregarina spp.; Pneumocystis carinii; membersof the Microspora phylum such as, for example, Nosema, Enterocytozoon,Encephalitozoon, Septata, Mrazekia, Amblyospora, Arneson, Glugea,Pleistophora and Microsporidium spp.; and members of the Ascetosporaphylum such as, for example, Haplosporidium spp as well as speciesincluding Plasmodium falciparum, P. vivax, P. ovale, P. malaria;Toxoplasma gondii; Leishmania mexicana, L. tropica, L. major, L.aethiopica, L. donovani, Trypanosoma cruzi, T brucei, Schistosomamansoni, S. haematobium, S. japonium; Trichinella spiralis; Wuchereriabancrofti; Brugia malayli; Entamoeba histolytica; Enterobiusvermiculoarus; Taenia solium, T saginata, Trichomonas vaginatis, T.hominis, T. tenax; Giardia lamblia; Cryptosporidium parvum; Pneumocytiscarinii, Babesia bovis, B. divergens, B. microti, Isospora belli, Lhominis; Dientamoeba fragilis; Onchocerca volvulus; Ascarislumbricoides; Necator americanis; Ancylostoma duodenale; Strongyloidesstercotalis; Capillaria philippinensis; Angiostrongylus cantonensis;Hymenolepis nana; Diphyllobothrium latum; Echinococcus granulosus, E.multilocularis; Paragonimus westermani, P. caliensis; Chlonorchissinensis; Opisthorchis felineas, G. Viverini, Fasciola hepatica,Sarcoptes scabiei, Pediculus humanus; Phthirlus pubis; and Dermatobiahominis, as well as any other parasite now known or later identified tobe pathogenic.

5.4.2 Insertion of the Heterologous Gene Sequence

Insertion of a foreign gene sequence into a viral vector of theinvention can be accomplished by either a complete replacement of aviral coding region with a heterologous sequence or by a partialreplacement or by adding the heterologous nucleotide sequence to theviral genome. Complete replacement would probably best be accomplishedthrough the use of PCR-directed mutagenesis. Briefly, PCR-primer A wouldcontain, from the 5′ to 3′ end: a unique restriction enzyme site, suchas a class IIS restriction enzyme site (i.e., a “shifter” enzyme; thatrecognizes a specific sequence but cleaves the DNA either upstream ordownstream of that sequence); a stretch of nucleotides complementary toa region of the gene that is to be replaced; and a stretch ofnucleotides complementary to the carboxy-terminus coding portion of theheterologous sequence. PCR-primer B would contain from the 5′ to 3′ end:a unique restriction enzyme site; a stretch of nucleotides complementaryto the gene that is to be replaced; and a stretch of nucleotidescorresponding to the 5′ coding portion of the heterologous or non-nativegene. After a PCR reaction using these primers with a cloned copy of theheterologous or non-native gene, the product may be excised and clonedusing the unique restriction sites. Digestion with the class IIS enzymeand transcription with the purified phage polymerase would generate aRNA molecule containing the exact untranslated ends of the viral genethat carries now a heterologous or non-native gene insertion. In analternate embodiment, PCR-primed reactions could be used to preparedouble-stranded DNA containing the bacteriophage promoter sequence, andthe hybrid gene sequence so that RNA templates can be transcribeddirectly without cloning.

A heterologous nucleotide sequence can be added or inserted at variouspositions of the virus of the invention. In one embodiment, theheterologous nucleotide sequence is added or inserted at position 1. Inanother embodiment, the heterologous nucleotide sequence is added orinserted at position 2. In another embodiment, the heterologousnucleotide sequence is added or inserted at position 3. In anotherembodiment, the heterologous nucleotide sequence is added or inserted atposition 4. In another embodiment, the heterologous nucleotide sequenceis added or inserted at position 5. In yet another embodiment, theheterologous nucleotide sequence is added or inserted at position 6. Asused herein, the term “position” refers to the position of theheterologous nucleotide sequence on the viral genome to be transcribed,e.g., position 1 means that it is the first gene to be transcribed, andposition 2 means that it is the second gene to be transcribed. Insertingheterologous nucleotide sequences at the lower-numbered positions of thevirus generally results in stronger expression of the heterologousnucleotide sequence compared to insertion at higher-numbered positionsdue to a transcriptional gradient that occurs across the genome of thevirus. However, the transcriptional gradient also yields specific ratiosof viral mRNAs. Insertion of foreign genes will perturb these ratios andresult in the synthesis of different amounts of viral proteins that mayinfluence virus replication. Thus, both the transcriptional gradient andthe replication kinetics must be considered when choosing an insertionsite. Inserting heterologous nucleotide sequences at lower-numberedpositions is the preferred embodiment of the invention if strongexpression of the heterologous nucleotide sequence is desired. In apreferred embodiment, the heterologous sequence is added or inserted atposition 1, 2 or 3.

When inserting a heterologous nucleotide sequence into the virus of theinvention, the intergenic region between the end of the coding sequenceof the heterologous gene and the start of the coding sequence of thedownstream gene can be altered to achieve a desired effect. As usedherein, the term “intergenic region” refers to nucleotide sequencebetween the stop signal of one gene and the start codon (e.g., AUG) ofthe coding sequence of the next downstream open reading frame. Anintergenic region may comprise a non-coding region of a gene, i.e.,between the transcription start site and the start of the codingsequence (AUG) of the gene. This non-coding region occurs naturally insome viral genes.

In various embodiments, the intergenic region between the heterologousnucleotide sequence and the downstream gene can be engineered,independently from each other, to be at least 10 nt in length, at least20 nt in length, at least 30 nt in length, at least 50 nt in length, atleast 75 nt in length, at least 100 nt in length, at least 125 nt inlength, at least 150 nt in length, at least 175 nt in length or at least200 nt in length. In certain embodiments, the intergenic region betweenthe heterologous nucleotide sequence and the downstream gene can beengineered, independently from each other, to be at most 10 nt inlength, at most 20 nt in length, at most 30 nt in length, at most 50 ntin length, at most 75 nt in length, at most 100 nt in length, at most125 nt in length, at most 150 nt in length, at most 175 nt in length orat most 200 nt in length. In various embodiments, the non-coding regionof a desired gene in a virus genome can also be engineered,independently from each other, to be at least 10 nt in length, at least20 nt in length, at least 30 nt in length, at least 50 nt in length, atleast 75 nt in length, at least 100 nt in length, at least 125 nt inlength, at least 150 nt in length, at least 175 nt in length or at least200 nt in length. In certain embodiments, the non-coding region of adesired gene in a virus genome can also be engineered, independentlyfrom each other, to be at most 10 nt in length, at most 20 nt in length,at most 30 nt in length, at most 50 nt in length, at most 75 nt inlength, at most 100 nt in length, at most 125 nt in length, at most 150nt in length, at most 175 nt in length or at most 200 nt in length.

When inserting a heterologous nucleotide sequence, the positional effectand the intergenic region manipulation can be used in combination toachieve a desirable effect. For example, the heterologous nucleotidesequence can be added or inserted at a position selected from the groupconsisting of position 1, 2, 3, 4, 5, and 6, and the intergenic regionbetween the heterologous nucleotide sequence and the next downstreamgene can be altered (see Table 3). Some of the combinations encompassedby the present invention are shown by way of example in Table 3.

TABLE 3 Examples of mode of insertion of heterologous nucleotidesequences Position 1 Position 2 Position 3 Position 4 Position 5Position 6 IGR^(a) 10-20 10-20 10-20 10-20 10-20 10-20 IGR 21-40 21-4021-40 21-40 21-40 21-40 IGR 41-60 41-60 41-60 41-60 41-60 41-60 IGR61-80 61-80 61-80 61-80 61-80 61-80 IGR  81-100  81-100  81-100  81-100 81-100  81-100 IGR 101-120 101-120 101-120 101-120 101-120 101-120 IGR121-140 121-140 121-140 121-140 121-140 121-140 IGR 141-160 141-160141-160 141-160 141-160 141-160 IGR 161-180 161-180 161-180 161-180161-180 161-180 IGR 181-200 181-200 181-200 181-200 181-200 181-200 IGR201-220 201-220 201-220 201-220 201-220 201-220 IGR 221-240 221-240221-240 221-240 221-240 221-240 IGR 241-260 241-260 241-260 241-260241-260 241-260 IGR 261-280 261-280 261-280 261-280 261-280 261-280 IGR281-300 281-300 281-300 281-300 281-300 281-300 ^(a)Intergenic Region,measured in nucleotide.

Depending on the purpose (e.g., to have strong immunogenicity) of theinserted heterologous nucleotide sequence, the position of the insertionand the length of the intergenic region of the inserted heterologousnucleotide sequence can be determined by various indexes including, butnot limited to, replication kinetics and protein or mRNA expressionlevels, measured by following non-limiting examples of assays: plaqueassay, fluorescent-focus assay, infectious center assay, transformationassay, endpoint dilution assay, efficiency of plating, electronmicroscopy, hemagglutination, measurement of viral enzyme activity,viral neutralization, hemagglutination inhibition, complement fixation,immunostaining, immunoprecipitation and immunoblotting, enzyme-linkedimmunosorbent assay, nucleic acid detection (e.g., Southern blotanalysis, Northern blot analysis, Western blot analysis), growth curve,employment of a reporter gene (e.g., using a reporter gene, such asGreen Fluorescence Protein (GFP) or enhanced Green Fluorescence Protein(eGFP), integrated to the viral genome the same fashion as theinterested heterologous gene to observe the protein expression), or acombination thereof. Procedures of performing these assays are wellknown in the art (see, e.g., Flint et al., PRINCIPLES OF VIROLOGY,MOLECULAR BIOLOGY, PATHOGENESIS, AND CONTROL, 2000, ASM Press pp. 25-56,the entire text is incorporated herein by reference), and non-limitingexamples are given in the Example sections, infra.

For example, expression levels can be determined by infecting cells inculture with a virus of the invention and subsequently measuring thelevel of protein expression by, e.g., Western blot analysis or ELISAusing antibodies specific to the gene product of the heterologoussequence, or measuring the level of RNA expression by, e.g., Northernblot analysis using probes specific to the heterologous sequence.Similarly, expression levels of the heterologous sequence can bedetermined by infecting an animal model and measuring the level ofprotein expressed from the heterologous sequence of the recombinantvirus of the invention in the animal model. The protein level can bemeasured by obtaining a tissue sample from the infected animal and thensubjecting the tissue sample to Western blot analysis or ELISA, usingantibodies specific to the gene product of the heterologous sequence.Further, if an animal model is used, the titer of antibodies produced bythe animal against the gene product of the heterologous sequence can bedetermined by any technique known to the skilled artisan, including butnot limited to, ELISA.

As the heterologous sequences can be homologous to a nucleotide sequencein the genome of the virus, care should be taken that the probes and theantibodies are indeed specific to the heterologous sequence or its geneproduct.

In certain specific embodiments, expression levels of F-protein of hMPVfrom chimeric avian-human metapneumovirus can be determined by anytechnique known to the skilled artisan. Expression levels of theF-protein can be determined by infecting cells in a culture with thechimeric virus of the invention and measuring the level of proteinexpression by, e.g., Western blot analysis or ELISA using antibodiesspecific to the F-protein and/or the G-protein of hMPV, or measuring thelevel of RNA expression by, e.g., Northern blot analysis using probesspecific to the F-gene and/or the G-gene of human metapneumovirus.Similarly, expression levels of the heterologous sequence can bedetermined using an animal model by infecting an animal and measuringthe level of F-protein and/or G-protein in the animal model. The proteinlevel can be measured by obtaining a tissue sample from the infectedanimal and then subjecting the tissue sample to Western blot analysis orELISA using antibodies specific to F-protein and/or G-protein of theheterologous sequence. Further, if an animal model is used, the titer ofantibodies produced by the animal against F-protein and/or G-protein canbe determined by any technique known to the skilled artisan, includingbut not limited to, ELISA.

The rate of replication of a recombinant virus of the invention can bedetermined by any technique known to the skilled artisan.

In certain embodiments, to facilitate the identification of the optimalposition of the heterologous sequence in the viral genome and theoptimal length of the intergenic region, the heterologous sequenceencodes a reporter gene. Once the optimal parameters are determined, thereporter gene is replaced by a heterologous nucleotide sequence encodingan antigen of choice. Any reporter gene known to the skilled artisan canbe used with the methods of the invention. For more detail, see section5.8.

The rate of replication of the recombinant virus can be determined byany standard technique known to the skilled artisan. The rate ofreplication is represented by the growth rate of the virus and can bedetermined by plotting the viral titer over the time post infection. Theviral titer can be measured by any technique known to the skilledartisan. In certain embodiments, a suspension containing the virus isincubated with cells that are susceptible to infection by the virus.Cell types that can be used with the methods of the invention include,but are not limited to, Vero cells, LLC-MK-2 cells, Hep-2 cells, LF 1043(HEL) cells, MRC-5 cells, WI-38 cells, tMK cells, 293 T cells, QT 6cells, QT 35 cells, or chicken embryo fibroblasts (CEF). Subsequent tothe incubation of the virus with the cells, the number of infected cellsis determined. In certain specific embodiments, the virus comprises areporter gene. Thus, the number of cells expressing the reporter gene isrepresentative of the number of infected cells. In a specificembodiment, the virus comprises a heterologous nucleotide sequenceencoding for eGFP, and the number of cells expressing eGFP, i.e., thenumber of cells infected with the virus, is determined using FACS.

In certain embodiments, the replication rate of the recombinant virus ofthe invention is at most 20% of the replication rate of the wild typevirus from which the recombinant virus is derived under the sameconditions. The same conditions refer to the same initial titer ofvirus, the same strain of cells, the same incubation temperature, growthmedium, number of cells and other test conditions that may affect thereplication rate. For example, the replication rate of APV/hMPV withPIV's F gene in position 1 is at most 20% of the replication rate ofAPV.

In certain embodiments, the replication rate of the recombinant virus ofthe invention is at most 5%, at most 10%, at most 20%, at most 30%, atmost 40%, at most 50%, at most 75%, at most 80%, at most 90% of thereplication rate of the wild type virus from which the recombinant virusis derived under the same conditions. In certain embodiments, thereplication rate of the recombinant virus of the invention is at least5%, at least 10%, at least 20%, at least 30%, at least 40%, at least50%, at least 75%, at least 80%, at least 90% of the replication rate ofthe wild type virus from which the recombinant virus is derived underthe same conditions. In certain embodiments, the replication rate of therecombinant virus of the invention is between 5% and 20%, between 10%and 40%, between 25% and 50%, between 40% and 75%, between 50% and 80%,or between 75% and 90% of the replication rate of the wild type virusfrom which the recombinant virus is derived under the same conditions.

In certain embodiments, the expression level of the heterologoussequence in the recombinant virus of the invention is at most 20% of theexpression level of the F-protein of the wild type virus from which therecombinant virus is derived under the same conditions. The sameconditions refer to the same initial titer of virus, the same strain ofcells, the same incubation temperature, growth medium, number of cellsand other test conditions that may affect the replication rate. Forexample, the expression level of the heterologous sequence of theF-protein of PIV3 in position 1 of hMPV is at most 20% of the expressionlevel of the F-protein of hMPV.

In certain embodiments, the expression level of the heterologoussequence in the recombinant virus of the invention is at most 5%, atmost 10%, at most 20%, at most 30%, at most 40%, at most 50%, at most75%, at most 80%, at most 90% of the expression level of the F-proteinof the wild type virus from which the recombinant virus is derived underthe same conditions. In certain embodiments, the expression level of theheterologous sequence in the recombinant virus of the invention is atleast 5%, at least 10%, at least 20%, at least 30%, at least 40%, atleast 50%, at least 75%, at least 80%, at least 90% of the expressionlevel of the F-protein of the wild type virus from which the recombinantvirus is derived under the same conditions. In certain embodiments, theexpression level of the heterologous sequence in the recombinant virusof the invention is between 5% and 20%, between 10% and 40%, between 25%and 50%, between 40% and 75%, between 50% and 80%, or between 75% and90% of the expression level of the F-protein of the wild type virus fromwhich the recombinant virus is derived under the same conditions.

5.4.3 Insertion of the Heterologous Gene Sequence into the G Gene

The G protein is a transmembrane protein of metapneumoviruses. In aspecific embodiment, the heterologous sequence is inserted into theregion of the G-ORF that encodes for the ectodomain, such that it isexpressed on the surface of the viral envelope. In one approach, theheterologous sequence may be inserted within the antigenic site withoutdeleting any viral sequences. In another approach, the heterologoussequences replaces sequences of the G-ORF. Expression products of suchconstructs may be useful in vaccines against the foreign antigen, andmay indeed circumvent problems associated with propagation of therecombinant virus in the vaccinated host. An intact G molecule with asubstitution only in antigenic sites may allow for G function and thusallow for the construction of a viable virus. Therefore, this virus canbe grown without the need for additional helper functions. The virus mayalso be attenuated in other ways to avoid any danger of accidentalescape.

Other hybrid constructions may be made to express proteins on the cellsurface or enable them to be released from the cell.

5.4.4 Construction of Bicistronic RNA

Bicistronic mRNA could be constructed to permit internal initiation oftranslation of viral sequences and allow for the expression of foreignprotein coding sequences from the regular terminal initiation site.Alternatively, a bicistronic mRNA sequence may be constructed whereinthe viral sequence is translated from the regular terminal open readingframe, while the foreign sequence is initiated from an internal site.Certain internal ribosome entry site (IRES) sequences may be utilized.The IRES sequences which are chosen should be short enough to notinterfere with MPV packaging limitations. Thus, it is preferable thatthe IRES chosen for such a bicistronic approach be no more than 500nucleotides in length. In a specific embodiment, the IRES is derivedfrom a picornavirus and does not include any additional picornaviralsequences. Specific IRES elements include, but are not limited to themammalian BiP IRES and the hepatitis C virus IRES.

Alternatively, a foreign protein may be expressed from a new internaltranscriptional unit in which the transcriptional unit has an initiationsite and polyadenylation site. In another embodiment, the foreign geneis inserted into a MPV gene such that the resulting expressed protein isa fusion protein.

5.5 Expression of Heterologous Gene Products Using Recombinant cDNA andrNA Templates

The viral vectors and recombinant templates prepared as described abovecan be used in a variety of ways to express the heterologous geneproducts in appropriate host cells or to create chimeric viruses thatexpress the heterologous gene products. In one embodiment, therecombinant cDNA can be used to transfect appropriate host cells and theresulting RNA may direct the expression of the heterologous gene productat high levels. Host cell systems which provide for high levels ofexpression include continuous cell lines that supply viral functionssuch as cell lines superinfected with APV or MPV, respectively, celllines engineered to complement APV or MPV functions, etc.

In an alternate embodiment of the invention, the recombinant templatesmay be used to transfect cell lines that express a viral polymeraseprotein in order to achieve expression of the heterologous gene product.To this end, transformed cell lines that express a polymerase proteinsuch as the L protein may be utilized as appropriate host cells. Hostcells may be similarly engineered to provide other viral functions oradditional functions such as G or N.

In another embodiment, a helper virus may provide the RNA polymeraseprotein utilized by the cells in order to achieve expression of theheterologous gene product. In yet another embodiment, cells may betransfected with vectors encoding viral proteins such as the N, P, L,and M2-1 proteins.

5.6 Rescue of Recombinant Virus Particles

In order to prepare the chimeric and recombinant viruses of theinvention, a cDNA encoding the genome of a recombinant or chimeric virusof the invention in the plus or minus sense may be used to transfectcells which provide viral proteins and functions required forreplication and rescue. Alternatively, cells may be transfected withhelper virus before, during, or after transfection by the DNA or RNAmolecule coding for the recombinant virus of the invention. Thesynthetic recombinant plasmid DNAs and RNAs of the invention can bereplicated and rescued into infectious virus particles by any number oftechniques known in the art, as described, e.g., in U.S. Pat. No.5,166,057 issued Nov. 24, 1992; in U.S. Pat. No. 5,854,037 issued Dec.29, 1998; in European Patent Publication EP 0702085A1, published Feb.20, 1996; in U.S. patent application Ser. No. 09/152,845; inInternational Patent Publications PCT WO97/12032 published Apr. 3, 1997;WO96/34625 published Nov. 7, 1996; in European Patent PublicationEP-A780475; WO 99/02657 published Jan. 21, 1999; WO 98/53078 publishedNov. 26, 1998; WO 98/02530 published Jan. 22, 1998; WO 99/15672published Apr. 1, 1999; WO 98/13501 published Apr. 2, 1998; WO 97/06270published Feb. 20, 1997; and EPO 780 47SA1 published Jun. 25, 1997, eachof which is incorporated by reference herein in its entirety.

In one embodiment, of the present invention, synthetic recombinant viralRNAs may be prepared that contain the non-coding regions of the negativestrand virus RNA which are essential for the recognition by viralpolymerases and for packaging signals necessary to generate a maturevirion. There are a number of different approaches which may be used toapply the reverse genetics approach to rescue negative strand RNAviruses. First, the recombinant RNAs are synthesized from a recombinantDNA template and reconstituted in vitro with purified viral polymerasecomplex to form recombinant ribonucleoproteins (RNPs) which can be usedto transfect cells. In another approach, a more efficient transfectionis achieved if the viral polymerase proteins are present duringtranscription of the synthetic RNAs either in vitro or in vivo. Withthis approach the synthetic RNAs may be transcribed from cDNA plasmidswhich are either co-transcribed in vitro with cDNA plasmids encoding thepolymerase proteins, or transcribed in vivo in the presence ofpolymerase proteins, i.e., in cells which transiently or constitutivelyexpress the polymerase proteins.

In additional approaches described herein, the production of infectiouschimeric or recombinant virus may be replicated in host cell systemsthat express a metapneumoviral polymerase protein (e.g., in virus/hostcell expression systems; transformed cell lines engineered to express apolymerase protein, etc.), so that infectious chimeric or recombinantvirus are rescued. In this instance, helper virus need not be utilizedsince this function is provided by the viral polymerase proteinsexpressed.

In accordance with the present invention, any technique known to thoseof skill in the art may be used to achieve replication and rescue ofrecombinant and chimeric viruses. One approach involves supplying viralproteins and functions required for replication in vitro prior totransfecting host cells. In such an embodiment, viral proteins may besupplied in the form of wild-type virus, helper virus, purified viralproteins or recombinantly expressed viral proteins. The viral proteinsmay be supplied prior to, during or post transcription of the syntheticcDNAs or RNAs encoding the chimeric virus. The entire mixture may beused to transfect host cells. In another approach, viral proteins andfunctions required for replication may be supplied prior to or duringtranscription of the synthetic cDNAs or RNAs encoding the chimericvirus. In such an embodiment, viral proteins and functions required forreplication are supplied in the form of wild-type virus, helper virus,viral extracts, synthetic cDNAs or RNAs which express the viral proteinsare introduced into the host cell via infection or transfection. Thisinfection/transfection takes place prior to or simultaneous to theintroduction of the synthetic cDNAs or RNAs encoding the chimeric virus.

In a particularly desirable approach, cells engineered to express allviral genes or chimeric or recombinant virus of the invention, i.e.,APV, MPV, MPV/APV or APV/MPV, may result in the production of infectiousvirus which contain the desired genotype; thus eliminating the need fora selection system. Theoretically, one can replace any one of the ORFsor part of any one of the ORFs encoding structural proteins of MPV witha foreign sequence. However, a necessary part of this equation is theability to propagate the defective virus (defective because a normalviral gene product is missing or altered). A number of possibleapproaches exist to circumvent this problem. In one approach a virushaving a mutant protein can be grown in cell lines which are constructedto constitutively express the wild type version of the same protein. Bythis way, the cell line complements the mutation in the virus. Similartechniques may be used to construct transformed cell lines thatconstitutively express any of the MPV genes. These cell lines which aremade to express the viral protein may be used to complement the defectin the chimeric or recombinant virus and thereby propagate it.Alternatively, certain natural host range systems may be available topropagate chimeric or recombinant virus.

In yet another embodiment, viral proteins and functions required forreplication may be supplied as genetic material in the form of syntheticcDNAs or RNAs so that they are co-transcribed with the synthetic cDNAsor RNAs encoding the chimeric virus. In a particularly desirableapproach, plasmids which express the chimeric virus and the viralpolymerase and/or other viral functions are co-transfected into hostcells. For example, plasmids encoding the genomic or antigenomic APV,MPV, MPV/APV or APV/MPV RNA, with or without one or more heterologoussequences, may be co-transfected into host cells with plasmids encodingthe metapneumoviral polymerase proteins N, P, L, or M2-1. Alternatively,rescue of the recombinant viruses of the invention may be accomplishedby the use of Modified Vaccinia Virus Ankara (MVA) encoding T7 RNApolymerase, or a combination of MVA and plasmids encoding the polymeraseproteins (N, P, and L). For example, MVA-T7 or Fowl Pox-T7 can beinfected into Vero cells, LLC-MK-2 cells, Hep-2 cells, LF 1043 (HEL)cells, tMK cells, LLC-MK2, HUT 292, FRHL-2 (rhesus), FCL-1 (greenmonkey), WI-38 (human), MRC-5 (human) cells, 293 T cells, QT 6 cells, QT35 cells and CEF cells. After infection with MVA-T7 or Fowl Pox-T7, afull length antigenomic cDNA encoding the recombinant virus of theinvention may be transfected into the cells together with the N, P, L,and M2-1 encoding expression plasmids. Alternatively, the polymerase maybe provided by plasmid transfection. The cells and cell supernatant cansubsequently be harvested and subjected to a single freeze-thaw cycle.The resulting cell lysate may then be used to infect a fresh HeLa orVero cell monolayer in the presence of 1-beta-D-arabinofuranosylcytosine(ara C), a replication inhibitor of vaccinia virus, to generate a virusstock. The supernatant and cells from these plates can then beharvested, freeze-thawed once and the presence of recombinant virusparticles of the invention can be assayed by immunostaining of virusplaques using antiserum specific to the particular virus.

Another approach to propagating the chimeric or recombinant virus mayinvolve co-cultivation with wild-type virus. This could be done bysimply taking recombinant virus and co-infecting cells with this andanother wild-type virus. The wild-type virus should complement for thedefective virus gene product and allow growth of both the wild-type andrecombinant virus. Alternatively, a helper virus may be used to supportpropagation of the recombinant virus.

In another approach, synthetic templates may be replicated in cellsco-infected with recombinant viruses that express the metapneumoviruspolymerase protein. In fact, this method may be used to rescuerecombinant infectious virus in accordance with the invention. To thisend, the metapneumovirus polymerase protein may be expressed in anyexpression vector/host cell system, including but not limited to viralexpression vectors (e.g., vaccinia virus, adenovirus, baculovirus, etc.)or cell lines that express a polymerase protein (e.g., see Krystal etal., 1986, Proc. Natl. Acad. Sci. USA 83: 2709-2713). Moreover,infection of host cells expressing all metapneumovirus proteins mayresult in the production of infectious chimeric virus particles. Itshould be noted that it may be possible to construct a recombinant viruswithout altering virus viability. These altered viruses would then begrowth competent and would not need helper functions to replicate.

Transfection procedures are well-known to the skill artisan and include,but are not limited to, DEAE-dextran-mediated, Calciumphosphate-mediated, Electroporation, and Liposome-mediated transfection.

A full-length viral genome can be assembled from several smaller PCRfragments. Restriction maps of different isolates of hMPV are shown inFIG. 28. The restriction sites can be used to assemble the full-lengthconstruct. In certain embodiments, PCR primers are designed such thatthe fragment resulting from the PCR reaction has a restriction siteclose to its 5′ end and a restriction site close to it 3′ end. The PCRproduct can then be digested with the respective restriction enzymes andsubsequently ligated to the neighboring PCR fragments.

5.7 Attenuation of Recombinant Viruses

The recombinant viruses of the invention can be further geneticallyengineered to exhibit an attenuated phenotype. In particular, therecombinant viruses of the invention exhibit an attenuated phenotype ina subject to which the virus is administered as a vaccine. Attenuationcan be achieved by any method known to a skilled artisan. Without beingbound by theory, the attenuated phenotype of the recombinant virus canbe caused, e.g., by using a virus that naturally does not replicate wellin an intended host (e.g., using an APV in human), by reducedreplication of the viral genome, by reduced ability of the virus toinfect a host cell, or by reduced ability of the viral proteins toassemble to an infectious viral particle relative to the wild typestrain of the virus. The viability of certain sequences of the virus,such as the leader and the trailer sequence can be tested using aminigenome assay (see section 5.8).

The attenuated phenotypes of a recombinant virus of the invention can betested by any method known to the artisan (see, e.g., section 5.8). Acandidate virus can, for example, be tested for its ability to infect ahost or for the rate of replication in a cell culture system. In certainembodiments, a mini-genome system is used to test the attenuated viruswhen the gene that is altered is N, P, L, M2, F, G, M2-1, M2-2 or acombination thereof. In certain embodiments, growth curves at differenttemperatures are used to test the attenuated phenotype of the virus. Forexample, an attenuated virus is able to grow at 35° C., but not at 39°C. or 40° C. In certain embodiments, different cell lines can be used toevaluate the attenuated phenotype of the virus. For example, anattenuated virus may only be able to grow in monkey cell lines but notthe human cell lines, or the achievable virus titers in different celllines are different for the attenuated virus. In certain embodiments,viral replication in the respiratory tract of a small animal model,including but not limited to, hamsters, cotton rats, mice and guineapigs, is used to evaluate the attenuated phenotypes of the virus. Inother embodiments, the immune response induced by the virus, includingbut not limited to, the antibody titers (e.g., assayed by plaquereduction neutralization assay or ELISA) is used to evaluate theattenuated phenotypes of the virus. In a specific embodiment, the plaquereduction neutralization assay or ELISA is carried out at a low dose. Incertain embodiments, the ability of the recombinant virus to elicitpathological symptoms in an animal model can be tested. A reducedability of the virus to elicit pathological symptoms in an animal modelsystem is indicative of its attenuated phenotype. In a specificembodiment, the candidate viruses are tested in a monkey model for nasalinfection, indicated by mucous production.

The viruses of the invention can be attenuated such that one or more ofthe functional characteristics of the virus are impaired. In certainembodiments, attenuation is measured in comparison to the wild typestrain of the virus from which the attenuated virus is derived. In otherembodiments, attenuation is determined by comparing the growth of anattenuated virus in different host systems. Thus, for a non-limitingexample, an APV is said to be attenuated when grown in a human host ifthe growth of the APV in the human host is reduced compared to thegrowth of the APV in an avian host.

In certain embodiments, the attenuated virus of the invention is capableof infecting a host, is capable of replicating in a host such thatinfectious viral particles are produced. In comparison to the wild typestrain, however, the attenuated strain grows to lower titers or growsmore slowly. Any technique known to the skilled artisan can be used todetermine the growth curve of the attenuated virus and compare it to thegrowth curve of the wild type virus. For exemplary methods, see Examplesection, infra. In a specific embodiment, the attenuated virus grows toa titer of less than 10⁵ pfu/ml, of less than 10⁴ pfu/ml, of less than10³ pfu/ml, or of less than 10² pfu/ml in Vero cells under conditions asdescribed in, e.g., Example 22.

In certain embodiments, the attenuated virus of the invention (e.g., achimeric mammalian MPV) cannot replicate in human cells as well as thewild type virus (e.g., wild type mammalian MPV) does. However, theattenuated virus can replicate well in a cell line that lack interferonfunctions, such as Vero cells.

In other embodiments, the attenuated virus of the invention is capableof infecting a host, of replicating in the host, and of causing proteinsof the virus of the invention to be inserted into the cytoplasmicmembrane, but the attenuated virus does not cause the host to producenew infectious viral particles. In certain embodiments, the attenuatedvirus infects the host, replicates in the host, and causes viralproteins to be inserted in the cytoplasmic membrane of the host with thesame efficiency as the wild type mammalian virus. In other embodiments,the ability of the attenuated virus to cause viral proteins to beinserted into the cytoplasmic membrane into the host cell is reducedcompared to the wild type virus. In certain embodiments, the ability ofthe attenuated mammalian virus to replicate in the host is reducedcompared to the wild type virus. Any technique known to the skilledartisan can be used to determine whether a virus is capable of infectinga mammalian cell, of replicating within the host, and of causing viralproteins to be inserted into the cytoplasmic membrane of the host. Forillustrative methods see section 5.8.

In certain embodiments, the attenuated virus of the invention is capableof infecting a host. In contrast to the wild type mammalian MPV,however, the attenuated mammalian MPV cannot be replicated in the host.In a specific embodiment, the attenuated mammalian virus can infect ahost and can cause the host to insert viral proteins in its cytoplasmicmembranes, but the attenuated virus is incapable of being replicated inthe host. Any method known to the skilled artisan can be used to testwhether the attenuated mammalian MPV has infected the host and hascaused the host to insert viral proteins in its cytoplasmic membranes.

In certain embodiments, the ability of the attenuated mammalian virus toinfect a host is reduced compared to the ability of the wild type virusto infect the same host. Any technique known to the skilled artisan canbe used to determine whether a virus is capable of infecting a host. Forillustrative methods see section 5.8.

In certain embodiments, mutations (e.g., missense mutations) areintroduced into the genome of the virus to generate a virus with anattenuated phenotype. Mutations (e.g., missense mutations) can beintroduced into the N-gene, the P-gene, the M-gene, the F-gene, theM2-gene, the SH-gene, the G-gene or the L-gene of the recombinant virus.Mutations can be additions, substitutions, deletions, or combinationsthereof. In specific embodiments, a single amino acid deletion mutationfor the N, P, L, F, G, M2-1, M2-2 or M2 proteins is introduced, whichcan be screened for functionality in the mini-genome assay system and beevaluated for predicted functionality in the virus. In more specificembodiments, the missense mutation is a cold-sensitive mutation. Inother embodiments, the missense mutation is a heat-sensitive mutation.In one embodiment, major phosphorylation sites of P protein of the virusis removed. In another embodiment, a mutation or mutations areintroduced into the L gene of the virus to generate a temperaturesensitive strain. In yet another embodiment, the cleavage site of the Fgene is mutated in such a way that cleavage does not occur or occurs atvery low efficiency.

In other embodiments, deletions are introduced into the genome of therecombinant virus. In more specific embodiments, a deletion can beintroduced into the N-gene, the P-gene, the M-gene, the F-gene, theM2-gene, the SH-gene, the G-gene or the L-gene of the recombinant virus.In specific embodiments, the deletion is in the M2-gene of therecombinant virus of the present invention. In other specificembodiments, the deletion is in the SH-gene of the recombinant virus ofthe present invention. In yet another specific embodiment, both theM2-gene and the SH-gene are deleted.

In certain embodiments, the intergenic region of the recombinant virusis altered. In one embodiment, the length of the intergenic region isaltered. In another embodiment, the intergenic regions are shuffled from5′ to 3′ end of the viral genome.

In other embodiments, the genome position of a gene or genes of therecombinant virus is changed. In one embodiment, the F or G gene ismoved to the 3′ end of the genome. In another embodiment, the N gene ismoved to the 5′ end of the genome.

In certain embodiments, attenuation of the virus is achieved byreplacing a gene of the wild type virus with a gene of a virus of adifferent species, of a different subgroup, or of a different variant.In illustrative embodiments, the N-gene, the P-gene, the M-gene, theF-gene, the M2-gene, the SH-gene, the G-gene or the L-gene of amammalian MPV is replaced with the N-gene, the P-gene, the M-gene, theF-gene, the M2-gene, the SH-gene, the G-gene or the L-gene,respectively, of an APV. In other illustrative embodiments, the N-gene,the P-gene, the M-gene, the F-gene, the M2-gene, the SH-gene, the G-geneor the L-gene of APV is replaced with the N-gene, the P-gene, theM-gene, the F-gene, the M2-gene, the SH-gene, the G-gene or the L-gene,respectively, of a mammalian MPV. In a preferred embodiment, attenuationof the virus is achieved by replacing one or more polymerase associatedgenes (e.g., N, P, L or M2) with genes of a virus of a differentspecies.

In certain embodiments, attenuation of the virus is achieved byreplacing one or more specific domains of a protein of the wild typevirus with domains derived from the corresponding protein of a virus ofa different species. In an illustrative embodiment, the ectodomain of aF protein of APV is replaced with an ectodomain of a F protein of amammalian MPV. In a preferred embodiment, one or more specific domainsof L, N, or P protein are replaced with domains derived fromcorresponding proteins of a virus of a different species. In certainother embodiments, attenuation of the virus is achieved by deleting oneor more specific domains of a protein of the wild type virus. In aspecific embodiment, the transmembrane domain of the F-protein isdeleted.

In certain embodiments of the invention, the leader and/or trailersequence of the recombinant virus of the invention can be modified toachieve an attenuated phenotype. In certain, more specific embodiments,the leader and/or trailer sequence is reduced in length relative to thewild type virus by at least 1 nucleotide, at least 2 nucleotides, atleast 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides orat least 6 nucleotides. In certain other, more specific embodiments, thesequence of the leader and/or trailer of the recombinant virus ismutated. In a specific embodiment, the leader and the trailer sequenceare 100% complementary to each other. In other embodiments, 1nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides,6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, or 10nucleotides are not complementary to each other where the remainingnucleotides of the leader and the trailer sequences are complementary toeach other. In certain embodiments, the non-complementary nucleotidesare identical to each other. In certain other embodiments, thenon-complementary nucleotides are different from each other. In otherembodiments, if the non-complementary nucleotide in the trailer ispurine, the corresponding nucleotide in the leader sequence is also apurine. In other embodiments, if the non-complementary nucleotide in thetrailer is pyrimidine, the corresponding nucleotide in the leadersequence is also a purine.

When a live attenuated vaccine is used, its safety must also beconsidered. The vaccine must not cause disease. Any techniques known inthe art that can make a vaccine safe may be used in the presentinvention. In addition to attenuation techniques, other techniques maybe used. One non-limiting example is to use a soluble heterologous genethat cannot be incorporated into the virion membrane. For example, asingle copy of the soluble RSV F gene, a version of the RSV gene lackingthe transmembrane and cytosolic domains, can be used. Since it cannot beincorporated into the virion membrane, the virus tropism is not expectedto change.

Various assays can be used to test the safety of a vaccine. See section5.8, infra. Particularly, sucrose gradients and neutralization assayscan be used to test the safety. A sucrose gradient assay can be used todetermine whether a heterologous protein is inserted in a virion. If theheterologous protein is inserted in the virion, the virion should betested for its ability to cause symptoms even if the parental straindoes not cause symptoms. Without being bound by theory, if theheterologous protein is incorporated in the virion, the virus may haveacquired new, possibly pathological, properties.

5.8 Assays for Use with the Invention

A number of assays may be employed in accordance with the presentinvention in order to determine the rate of growth of a chimeric orrecombinant virus in a cell culture system, an animal model system or ina subject. A number of assays may also be employed in accordance withthe present invention in order to determine the requirements of thechimeric and recombinant viruses to achieve infection, replication andpackaging of virions.

The assays described herein may be used to assay viral titer over timeto determine the growth characteristics of the virus. In a specificembodiment, the viral titer is determined by obtaining a sample from theinfected cells or the infected subject, preparing a serial dilution ofthe sample and infecting a monolayer of cells that are susceptible toinfection with the virus at a dilution of the virus that allows for theemergence of single plaques. The plaques can then be counted and theviral titer express as plaque forming units per milliliter of sample. Ina specific embodiment of the invention, the growth rate of a virus ofthe invention in a subject is estimated by the titer of antibodiesagainst the virus in the subject. Without being bound by theory, theantibody titer in the subject reflects not only the viral titer in thesubject but also the antigenicity. If the antigenicity of the virus isconstant, the increase of the antibody titer in the subject can be usedto determine the growth curve of the virus in the subject. In apreferred embodiment, the growth rate of the virus in animals or humansis best tested by sampling biological fluids of a host at multiple timepoints post-infection and measuring viral titer.

The expression of heterologous gene sequence in a cell culture system orin a subject can be determined by any technique known to the skilledartisan. In certain embodiments, the expression of the heterologous geneis measured by quantifying the level of the transcript. The level of thetranscript can be measured by Northern blot analysis or by RT-PCR usingprobes or primers, respectively, that are specific for the transcript.The transcript can be distinguished from the genome of the virus becausethe virus is in the antisense orientation whereas the transcript is inthe sense orientation. In certain embodiments, the expression of theheterologous gene is measured by quantifying the level of the proteinproduct of the heterologous gene. The level of the protein can bemeasured by Western blot analysis using antibodies that are specific tothe protein.

In a specific embodiment, the heterologous gene is tagged with a peptidetag. The peptide tag can be detected using antibodies against thepeptide tag. The level of peptide tag detected is representative for thelevel of protein expressed from the heterologous gene. Alternatively,the protein expressed from the heterologous gene can be isolated byvirtue of the peptide tag. The amount of the purified protein correlateswith the expression level of the heterologous gene. Such peptide tagsand methods for the isolation of proteins fused to such a peptide tagare well known in the art. A variety of peptide tags known in the artmay be used in the modification of the heterologous gene, such as, butnot limited to, the immunoglobulin constant regions, polyhistidinesequence (Petty, 1996, Metal-chelate affinity chromatography, in CurrentProtocols in Molecular Biology, volume 1-3 (1994-1998). Ed. by F. M.Ausubel, R. Brent, R. E. Kunston, D. D. Moore, J. G. Seidman, J. A.Smith, and K. Struhl, published by John Wiley and sons, Inc., USA,Greene Publish. Assoc. & Wiley Interscience), glutathione S-transferase(GST; Smith, 1993, Methods Mol. Cell. Bio. 4:220-229), the E. colimaltose binding protein (Guan et al., 1987, Gene 67:21-30), variouscellulose binding domains (U.S. Pat. Nos. 5,496,934; 5,202,247; U.S.Pat. No. 5,137,819; Tomme et al., 1994, Protein Eng. 7:117-123), and theFLAG epitope (Short Protocols in Molecular Biology, 1999, Ed. Ausubel etal., John Wiley & Sons, Inc., Unit 10.11) etc. Other peptide tags arerecognized by specific binding partners and thus facilitate isolation byaffinity binding to the binding partner, which is preferably immobilizedand/or on a solid support. As will be appreciated by those skilled inthe art, many methods can be used to obtain the coding region of theabove-mentioned peptide tags, including but not limited to, DNA cloning,DNA amplification, and synthetic methods. Some of the peptide tags andreagents for their detection and isolation are available commercially.

Samples from a subject can be obtained by any method known to theskilled artisan. In certain embodiments, the sample consists of nasalaspirate, throat swab, sputum or broncho-alveolar lavage.

5.8.1 Minireplicon Constructs

Minireplicon constructs can be generated to contain an antisensereporter gene. Any reporter gene known to the skilled artisan can beused with the invention (see section 5.8.2). In a specific embodiment,the reporter gene is CAT. In certain embodiments, the reporter gene canbe flanked by the negative-sense hMPV or APV leader linked to thehepatitis delta ribozyme (Hep-d Ribo) and T7 polymerase termination(T-T7) signals, and the hMPV or APV trailer sequence preceded by the T7RNA polymerase promoter.

In certain embodiments, the plasmid encoding the minireplicon istransfected into a host cell. The host cell expresses T7 RNA polymerase,the N gene, the P gene, the L gene, and the M2.1 gene. In certainembodiments, the host cell is transfected with plasmids encoding T7 RNApolymerase, the N gene, the P gene, the L gene, and the M2.1 gene. Inother embodiments, the plasmid encoding the minireplicon is transfectedinto a host cell and the host cell is infected with a helper virus.

The expression level of the reporter gene and/or its activity can beassayed by any method known to the skilled artisan, such as, but notlimited to, the methods described in section 5.8.2.

In certain, more specific, embodiments, the minireplicon comprises thefollowing elements, in the order listed: T7 RNA Polymerase or RNApolymerase I, leader sequence, gene start, GFP, trailer sequence,Hepatitis delta ribozyme sequence or RNA polymerase I terminationsequence. If T7 is used as RNA polymerase, Hepatitis delta ribozymesequence should be used as termination sequence. If RNA polymerase I isused, RNA polymerase I termination sequence may be used as a terminationsignal. Dependent on the rescue system, the sequence of the minirepliconcan be in the sense or antisense orientation. In certain embodiments,the leader sequence can be modified relative to the wild type leadersequence of hMPV. The leader sequence can optionally be preceded by anAC. The T7 promoter sequence can be with or without a G-doublet ortriplet, where the G-doublet or triplet provides for increasedtranscription.

In a specific embodiment, a cell is infected with hMPV at TO. 24 hourslater, at T24, the cell is transfected with a minireplicon construct. 48hours after TO and 72 hours after TO, the cells are tested for theexpression of the reporter gene. If a fluorescent reporter gene productis used (e.g., GFP), the expression of the reporter gene can be testedusing FACS.

In another embodiment, a cell is transfected with six plasmids at T=0hours. Cells are then harvested at T=40 hours and T50 hours and analyzedfor CAT or GFP expression. (See FIG. 25.)

In another specific embodiment, a cell is infected with MVA-T7 at TO. 1hour later, at Ti, the cell is transfected with a minirepliconconstruct. 24 hours after TO, the cell is infected with hMPV. 72 hoursafter TO, the cells are tested for the expression of the reporter gene.If a fluorescent reporter gene product is used (e.g., GFP), theexpression of the reporter gene can be tested using FACS.

5.8.2 Reporter Genes

In certain embodiments, assays for measurement of reporter geneexpression in tissue culture or in animal models can be used with themethods of the invention. The nucleotide sequence of the reporter geneis cloned into the virus, such as APV, hMPV, hMPV/APV or APV/hMPV,wherein (i) the position of the reporter gene is changed and (ii) thelength of the intergenic regions flanking the reporter gene are varied.Different combinations are tested to determine the optimal rate ofexpression of the reporter gene and the optimal replication rate of thevirus comprising the reporter gene.

In certain embodiments, minireplicon constructs are generated to includea reporter gene. The construction of minireplicon constructs isdescribed herein.

The abundance of the reporter gene product can be determined by anytechnique known to the skilled artisan. Such techniques include, but arenot limited to, Northern blot analysis or Western blot analysis usingprobes or antibodies, respectively, that are specific to the reportergene.

In certain embodiments, the reporter gene emits a fluorescent signalthat can be detected in a FACS. FACS can be used to detect cells inwhich the reporter gene is expressed.

Techniques for practicing the specific aspect of this invention willemploy, unless otherwise indicated, conventional techniques of molecularbiology, microbiology, and recombinant DNA manipulation and production,which are routinely practiced by one of skill in the art. See, e.g.,Sambrook et al., Molecular cloning, a laboratory manual, second ed.,vol. 1-3. (Cold Spring Harbor Laboratory, 1989), A Laboratory Manual,Second Edition; DNA Cloning, Volumes I and II (Glover, Ed. 1985); andTranscription and Translation (Hames & Higgins, Eds. 1984).

The biochemical activity of the reporter gene product represents theexpression level of the reporter gene. The total level of reporter geneactivity depends also on the replication rate of the recombinant virusof the invention. Thus, to determine the true expression level of thereporter gene from the recombinant virus, the total expression levelshould be divided by the titer of the recombinant virus in the cellculture or the animal model.

Reporter genes that can be used with the methods of invention include,but are not limited to, the genes listed in the Table 4 below:

TABLE 4 Reporter genes and the biochemical properties of the respectivereporter gene products Reporter Gene Protein Activity & Measurement CAT(chloramphenicol Transfers radioactive acetyl groups toacetyltransferase) chloramphenicol or detection by thin layerchromatography and autoradiography GAL (β-galactosidase) Hydrolyzescolorless galactosides to yield colored products. GUS (β-glucuronidase)Hydrolyzes colorless glucuronides to yield colored products. LUC(luciferase) Oxidizes luciferin, emitting photons GFP (green fluorescentfluorescent protein without substrate protein) SEAP (secretedluminescence reaction with suitable substrates alkaline phosphatase) orwith substrates that generate chromophores HRP (horseradish in thepresence of hydrogen oxide, oxidation peroxidase) of3,3′,5,5′-tetramethylbenzidine to form a colored complex AP (alkalineluminescence reaction with suitable substrates phosphatase) or withsubstrates that generate chromophores

The abundance of the reporter gene can be measured by, inter alia,Western blot analysis or Northern blot analysis or any other techniqueused for the quantification of transcription of a nucleotide sequence,the abundance of its mRNA its protein (see Short Protocols in MolecularBiology, Ausubel et al., (editors), John Wiley & Sons, Inc., 4^(th)edition, 1999). In certain embodiments, the activity of the reportergene product is measured as a readout of reporter gene expression fromthe recombinant virus. For the quantification of the activity of thereporter gene product, biochemical characteristics of the reporter geneproduct can be employed (see Table 4). The methods for measuring thebiochemical activity of the reporter gene products are well-known to theskilled artisan. A more detailed description of illustrative reportergenes that can be used with the methods of the invention is set forthbelow.

5.8.3 Measurement of Incidence of Infection Rate

The incidence of infection can be determined by any method well-known inthe art, for example, but not limited to, clinical samples (e.g., nasalswabs) can be tested for the presence of a virus of the invention byimmunofluorescence assay (IFA) using an anti-APV-antigen antibody, ananti-hMPV-antigen antibody, an anti-APV-antigen antibody, and/or anantibody that is specific to the gene product of the heterologousnucleotide sequence, respectively.

In certain embodiments, samples containing intact cells can be directlyprocessed, whereas isolates without intact cells should first becultured on a permissive cell line (e.g. HEp-2 cells). In anillustrative embodiments, cultured cell suspensions should be cleared bycentrifugation at, e.g., 300×g for 5 minutes at room temperature,followed by a PBS, pH 7.4 (Ca++ and Mg++ free) wash under the sameconditions. Cell pellets are resuspended in a small volume of PBS foranalysis. Primary clinical isolates containing intact cells are mixedwith PBS and centrifuged at 300×g for 5 minutes at room temperature.Mucus is removed from the interface with a sterile pipette tip and cellpellets are washed once more with PBS under the same conditions. Pelletsare then resuspended in a small volume of PBS for analysis. Five to tenmicroliters of each cell suspension are spotted per 5 mm well on acetonewashed 12-well HTC supercured glass slides and allowed to air dry.Slides are fixed in cold (−20° C.) acetone for 10 minutes. Reactions areblocked by adding PBS-1% BSA to each well followed by a 10 minuteincubation at room temperature. Slides are washed three times inPBS-0.1% TWEEN®-20 and air dried. Ten microliters of each primaryantibody reagent diluted to 250 ng/ml in blocking buffer is spotted perwell and reactions are incubated in a humidified 37° C. environment for30 minutes. Slides are then washed extensively in three changes ofPBS-0.1% TWEEN®-20 and air dried. Ten microliters of appropriatesecondary conjugated antibody reagent diluted to 250 ng/ml in blockingbuffer are spotted per respective well and reactions are incubated in ahumidified 37° C. environment for an additional 30 minutes. Slides arethen washed in three changes of PBS-0.1% TWEEN®-20. Five microliters ofPBS-50% glycerol-10 mM Tris pH 8.0-1 mM EDTA are spotted per reactionwell, and slides are mounted with cover slips. Each reaction well issubsequently analyzed by fluorescence microscopy at 200× power using aB-2A filter (EX 450-490 nm). Positive reactions are scored against anautofluorescent background obtained from unstained cells or cellsstained with secondary reagent alone. Positive reactions arecharacterized by bright fluorescence punctuated with small inclusions inthe cytoplasm of infected cells.

5.8.4 Measurement of Serum Titer

Antibody serum titer can be determined by any method well-known in theart, for example, but not limited to, the amount of antibody or antibodyfragment in serum samples can be quantitated by a sandwich ELISA.Briefly, the ELISA consists of coating microtiter plates overnight at 4°C. with an antibody that recognizes the antibody or antibody fragment inthe serum. The plates are then blocked for approximately 30 minutes atroom temperature with PBS-TWEEN®-0.5% BSA. Standard curves areconstructed using purified antibody or antibody fragment diluted inPBS-TWEEN®-BSA, and samples are diluted in PBS-BSA. The samples andstandards are added to duplicate wells of the assay plate and areincubated for approximately 1 hour at room temperature. Next, thenon-bound antibody is washed away with PBS-TWEEN® and the bound antibodyis treated with a labeled secondary antibody (e.g., horseradishperoxidase conjugated goat-anti-human IgG) for approximately 1 hour atroom temperature. Binding of the labeled antibody is detected by addinga chromogenic substrate specific for the label and measuring the rate ofsubstrate turnover, e.g., by a spectrophotometer. The concentration ofantibody or antibody fragment levels in the serum is determined bycomparison of the rate of substrate turnover for the samples to the rateof substrate turnover for the standard curve at a certain dilution.

5.8.5 Serological Tests

In certain embodiments of the invention, the presence of antibodies thatbind to a component of a mammalian MPV is detected. In particular thepresence of antibodies directed to a protein of a mammalian MPV can bedetected in a subject to diagnose the presence of a mammalian MPV in thesubject. Any method known to the skilled artisan can be used to detectthe presence of antibodies directed to a component of a mammalian MPV.

In an illustrative embodiment, components of mammalian MPV are linked toa solid support. In a specific embodiment, the component of themammalian MPV can be, but is not limited to, the F protein or the Gprotein. Subsequently, the material that is to be tested for thepresence of antibodies directed to mammalian MPV is incubated with thesolid support under conditions conducive to the binding of theantibodies to the mammalian MPV components. Subsequently, the solidsupport is washed under conditions that remove any unspecifically boundantibodies. Following the washing step, the presence of bound antibodiescan be detected using any technique known to the skilled artisan. In aspecific embodiment, the mammalian MPV protein-antibody complex isincubated with detectably labeled antibody that recognizes antibodiesthat were generated by the species of the subject, e.g., if the subjectis a cotton rat, the detectably labeled antibody is directed to ratantibodies, under conditions conducive to the binding of the detectablylabeled antibody to the antibody that is bound to the component ofmammalian MPV. In a specific embodiment, the detectably labeled antibodyis conjugated to an enzymatic activity. In another embodiment, thedetectably labeled antibody is radioactively labeled. The complex ofmammalian MPV protein-antibody-detectably labeled antibody is thenwashed, and subsequently the presence of the detectably labeled antibodyis quantified by any technique known to the skilled artisan, wherein thetechnique used is dependent on the type of label of the detectablylabeled antibody.

5.8.6 Biacore Assay

Determination of the kinetic parameters of antibody binding can bedetermined for example by the injection of 250 μl, of monoclonalantibody (“mAb”) at varying concentration in HBS buffer containing 0.05%TWEEN®-20 over a sensor chip surface, onto which has been immobilizedthe antigen. The antigen can be any component of a mammalian MPV. In aspecific embodiment, the antigen can be, but is not limited to, the Fprotein or the G protein of a mammalian MPV. The flow rate is maintainedconstant at 75 μL/min. Dissociation data is collected for 15 min, orlonger as necessary. Following each injection/dissociation cycle, thebound mAb is removed from the antigen surface using brief, 1-minutepulses of dilute acid, typically 10-100 mM HCl, though other regenerantsare employed as the circumstances warrant.

More specifically, for measurement of the rates of association, k_(on),and dissociation, k_(off), the antigen is directly immobilized onto thesensor chip surface through the use of standard amine couplingchemistries, namely the EDC/NHS method(EDC=N-diethylaminopropyl)-carbodiimide). Briefly, a 5-100 nM solutionof the antigen in 10 mM NaOAc, pH4 or pH5 is prepared and passed overthe EDC/NHS-activated surface until approximately 30-50 RU's (BiacoreResonance Unit) worth of antigen are immobilized. Following this, theunreacted active esters are “capped” off with an injection of 1M Et-NH2.A blank surface, containing no antigen, is prepared under identicalimmobilization conditions for reference purposes. Once a suitablesurface has been prepared, an appropriate dilution series of each one ofthe antibody reagents is prepared in HBS/TWEEN®-20, and passed over boththe antigen and reference cell surfaces, which are connected in series.The range of antibody concentrations that are prepared varies dependingon what the equilibrium binding constant, K_(D), is estimated to be. Asdescribed above, the bound antibody is removed after eachinjection/dissociation cycle using an appropriate regenerate.

Once an entire data set is collected, the resulting binding curves areglobally fitted using algorithms supplied by the instrumentmanufacturer, BIAcore, Inc. (Piscataway, N.J.). All data are fitted to a1:1 Langmuir binding model. These algorithm calculate both the k_(on)and the k_(off), from which the apparent equilibrium binding constant,K_(D), is deduced as the ratio of the two rate constants (i.e.,k_(off)/k_(on)). More detailed treatments of how the individual rateconstants are derived can be found in the BIAevaluation SoftwareHandbook (BIAcore, Inc., Piscataway, N.J.).

5.8.7 Microneutralization Assay

The ability of antibodies or antigen-binding fragments thereof toneutralize virus infectivity is determined by a microneutralizationassay. This microneutralization assay is a modification of theprocedures described by Anderson et al., (1985, J. Clin. Microbiol.22:1050-1052, the disclosure of which is hereby incorporated byreference in its entirety). The procedure is also described in Johnsonet al., 1999, J. Infectious Diseases 180:35-40, the disclosure of whichis hereby incorporated by reference in its entirety.

Antibody dilutions are made in triplicate using a 96-well plate. 10⁶TCID₅₀ of a mammalian MPV are incubated with serial dilutions of theantibody or antigen-binding fragments thereof to be tested for 2 hoursat 37° C. in the wells of a 96-well plate. Cells susceptible toinfection with a mammalian MPV, such as, but not limited to Vero cells(2.5×10⁴) are then added to each well and cultured for 5 days at 37° C.in 5% CO₂. After 5 days, the medium is aspirated and cells are washedand fixed to the plates with 80% methanol and 20% PBS. Virus replicationis then determined by viral antigen, such as F protein expression. Fixedcells are incubated with a biotin-conjugated anti-viral antigen, such asanti-F protein monoclonal antibody (e.g., pan F protein, C-site-specificMAb 133-1H) washed and horseradish peroxidase conjugated avidin is addedto the wells. The wells are washed again and turnover of substrate TMB(thionitrobenzoic acid) is measured at 450 nm. The neutralizing titer isexpressed as the antibody concentration that causes at least 50%reduction in absorbency at 450 nm (the OD₄₅₀) from virus-only controlcells.

The microneutralization assay described here is only one example.Alternatively, standard neutralization assays can be used to determinehow significantly the virus is affected by an antibody.

5.8.8 Viral Fusion Inhibition Assay

This assay is in principle identical to the microneutralization assay,except that the cells are infected with the respective virus for fourhours prior to addition of antibody and the read-out is in terms ofpresence of absence of fusion of cells (Taylor et al., 1992, J. Gen.Virol. 73:2217-2223).

5.8.9 Isothermal Titration Calorimetry

Thermodynamic binding affinities and enthalpies are determined fromisothermal titration calorimetry (ITC) measurements on the interactionof antibodies with their respective antigen.

Antibodies are diluted in dialysate and the concentrations weredetermined by UV spectroscopic absorption measurements with aPerkin-Elmer Lambda 4B Spectrophotometer using an extinction coefficientof 217,000 M⁻¹ cm⁻¹ at the peak maximum at 280 nm. The diluted mammalianMPV-antigen concentrations are calculated from the ratio of the mass ofthe original sample to that of the diluted sample since its extinctioncoefficient is too low to determine an accurate concentration withoutemploying and losing a large amount of sample.

ITC Measurements

The binding thermodynamics of the antibodies are determined from ITCmeasurements using a Microcal, Inc. VP Titration calorimeter. The VPtitration calorimeter consists of a matched pair of sample and referencevessels (1.409 ml) enclosed in an adiabatic enclosure and a rotatingstirrer-syringe for titrating ligand solutions into the sample vessel.The ITC measurements are performed at 25° C. and 35° C. The samplevessel contained the antibody in the phosphate buffer while thereference vessel contains just the buffer solution. The phosphate buffersolution is saline 67 mM PO₄ at pH 7.4 from HyClone, Inc. Five or tenaliquots of the 0.05 to 0.1 mM RSV-antigen, PIV-antigen, and/orhMPV-antigen solution are titrated 3 to 4 minutes apart into theantibody sample solution until the binding is saturated as evident bythe lack of a heat exchange signal.

A non-linear, least square minimization software program from Microcal,Inc., Origin 5.0, is used to fit the incremental heat of the i-thtitration (ΔQ(i)) of the total heat, Q_(t), to the total titrantconcentration, X_(t), according to the following equations (I),Q _(t) =nC _(t) ΔHb°V{1+X _(t) /nC _(t)+1/nK _(b) C _(t)−[(1+X _(t) /nC_(t)+1/nK _(b) C _(t))²−4X _(t) /nC _(t)]^(1/2)}/2  (1a)ΔQ(i)=Q(i)+dVi/2V{Q(i)+Q(i−1)}−Q(i−1)  (1b)where C_(t) is the initial antibody concentration in the sample vessel,V is the volume of the sample vessel, and n is the stoichiometry of thebinding reaction, to yield values of K_(b), ΔH_(b)°, and n. The optimumrange of sample concentrations for the determination of K_(b) depends onthe value of K_(b) and is defined by the following relationship.C _(t) K _(b) n≦0.500  (2)so that at 1 μM the maximum K_(b) that can be determined is less than2.5×10⁸ M⁻¹. If the first titrant addition does not fit the bindingisotherm, it was neglected in the final analysis since it may reflectrelease of an air bubble at the syringe opening-solution interface.

5.8.10 Immunoassays

Immunoprecipitation protocols generally comprise lysing a population ofcells in a lysis buffer such as RIPA buffer (I % NP-40 or TRITON® X-100,1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphateat pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/orprotease inhibitors (e.g., EDTA, PMSF, 159 aprotinin, sodium vanadate),adding the antibody of interest to the cell lysate, incubating for aperiod of time (e.g., to 4 hours) at 4 degrees C., adding protein Aand/or protein G sepharose beads to the cell lysate, incubating forabout an hour or more at 4 degrees C., washing the beads in lysis bufferand re-suspending the beads in SDS/sample buffer. The ability of theantibody of interest to immunoprecipitate a particular antigen can beassessed by, e.g., western blot analysis. One of skill in the art wouldbe knowledgeable as to the parameters that can be modified to increasethe binding of the antibody to an antigen and decrease the background(e.g., pre-clearing the cell lysate with sepharose beads). For furtherdiscussion regarding immunoprecipitation protocols see, e.g., Ausubel etal., eds., 1994, Current Protocols in Molecular Biology, Vol. 1, JohnWiley & Sons, Inc., New York at pages 10, 16, 1.

Western blot analysis generally comprises preparing protein samples,electrophoresis of the protein samples in a polyacrylamide gel (e.g.,8%-20% SDS-PAGE depending on the molecular weight of the antigen),transferring the protein sample from the polyacrylamide get to amembrane such as nitrocellulose, PVDF or nylon, blocking the membrane,in blocking solution (e.g., PBS with 3% BSA or non-fat milk), washingthe membrane in washing buffer (e.g., PBS TWEEN®-20), incubating themembrane with primary antibody (the antibody of interest) diluted inblocking buffer, washing the membrane in washing buffer, incubating themembrane with a secondary antibody (which recognizes the primaryantibody, e.g., an anti-human antibody) conjugated to an enzymaticsubstrate (e.g., horseradish peroxidase or alkaline phosphatase) orradioactive molecule (e.g., ¹²P or ¹²¹I) diluted in blocking buffer,washing the membrane in wash buffer, and detecting the presence of theantigen. One of skill in the art would be knowledgeable as to theparameters that can be modified to increase the signal detected and toreduce the background noise. For further discussion regarding westernblot protocols see, e.g., Ausubel et al., eds, 1994, GinTent Protocolsin Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at10.8.1.

ELISAs comprise preparing antigen, coating the well of a 96-wellmicrotiter plate with the antigen, washing away antigen that did notbind the wells, adding the antibody of interest conjugated to adetectable compound such as an enzymatic substrate (e.g., horseradishperoxidase or alkaline phosphatase) to the wells and incubating for aperiod of time, washing away unbound antibodies or non-specificallybound antibodies, and detecting the presence of the antibodiesspecifically bound to the antigen coating the well. In ELISAs theantibody of interest does not have to be conjugated to a detectablecompound; instead, a second antibody (which recognizes the antibody ofinterest) conjugated to a detectable compound may be added to the well.Further, instead of coating the well with the antigen, the antibody maybe coated to the well. In this case, the detectable molecule could bethe antigen conjugated to a detectable compound such as an enzymaticsubstrate (e.g., horseradish peroxidase or alkaline phosphatase). Theparameters that can be modified to increase signal detection and othervariations of ELISAs are well known to one of skill in the art. Forfurther discussion regarding ELISAs see, e.g., Ausubel et al., eds,1994, Current Protocols in Molecular Biology, Vol. I, John Wiley & Sons,Inc., New York at 11.2.1.

The binding affinity of an antibody (including a scFv or other moleculecomprising, or alternatively consisting of, antibody fragments orvariants thereof) to an antigen and the off-rate of an antibody-antigeninteraction can be determined by competitive binding assays. One exampleof a competitive binding assay is a radioimmunoassay comprising theincubation of labeled antigen (e.g., ³H or ¹²¹I) with the antibody ofinterest in the presence of increasing amounts of unlabeled antigen, andthe detection of the antibody bound to the labeled antigen.

5.8.11 Sucrose Gradient Assay

The question of whether the heterologous proteins are incorporated intothe virion can be further investigated by use of any biochemical assayknown to the skilled artisan. In a specific embodiment, a sucrosegradient assay is used to determine whether a heterologous protein isincorporated into the virion.

Infected cell lysates can be fractionated in 20-60% sucrose gradients,various fractions are collected and analyzed for the presence anddistribution of heterologous proteins and the vector proteins by, e.g.,Western blot analysis. The fractions and the virus proteins can also beassayed for peak virus titers by plaque assay. If the heterologousprotein co-migrates with the virion the heterologous protein isassociated with the virion.

5.9 Methods to Identify New Isolates of MPV

The present invention relates to mammalian MPV, in particular hMPV.While the present invention provides the characterization of twoserological subgroups of MPV, A and B, and the characterization of fourvariants of MPV A1, A2, B1 and B2, the invention is not limited to thesesubgroups and variants. The invention encompasses any yet to beidentified isolates of MPV, including those which are characterized asbelonging to the subgroups and variants described herein, or belongingto a yet to be characterized subgroup or variant.

Immunoassays can be used in order to characterize the protein componentsthat are present in a given sample. Immunoassays are an effective way tocompare viral isolates using peptides components of the viruses foridentification. For example, the invention provides herein a method toidentify further isolates of MPV as provided herein, the methodcomprising inoculating an essentially MPV-uninfected orspecific-pathogen-free guinea pig or ferret (in the detailed descriptionthe animal is inoculated intranasally but other was of inoculation suchas intramuscular or intradermal inoculation, and using anotherexperimental animal, is also feasible) with the prototype isolate 1-2614or related isolates. Sera are collected from the animal at day zero, twoweeks and three weeks post inoculation. The animal specificallyseroconverted as measured in virus neutralization (VN) assay (For anexample of a VN assay, see Example 16) and indirect IFA (For an exampleof IFA, see Example 11 or 14) against the respective isolate 1-2614 andthe sera from the seroconverted animal are used in the immunologicaldetection of the further isolates. As an example, the invention providesthe characterization of a new member in the family of Paramyxoviridae, ahuman metapneumovirus or metapneumovirus-like virus (since its finaltaxonomy awaits discussion by a viral taxonomy committee the MPV isherein for example described as taxonomically corresponding to APV)(MPV) which may cause severe RTI in humans. The clinical signs of thedisease caused by MPV are essentially similar to those caused by hRSV,such as cough, myalgia, vomiting, fever bronchiolitis or pneumonia,possible conjunctivitis, or combinations thereof. As is seen with hRSVinfected children, specifically very young children may requirehospitalization. As an example an MPV which was deposited Jan. 19, 2001as 1-2614 with CNCM, Institute Pasteur, Paris or a virus isolatephylogenetically corresponding therewith is herewith provided.Therewith, the invention provides a virus comprising a nucleic acid orfunctional fragment phylogenetically corresponding to a nucleic acidsequence of SEQ. ID NO:19, or structurally corresponding therewith. Inparticular the invention provides a virus characterized in that aftertesting it in phylogenetic tree analysis wherein maximum likelihoodtrees are generated using 100 bootstraps and 3 jumbles it is found to bemore closely phylogenetically corresponding to a virus isolate depositedas 1-2614 with CNCM, Paris than it is related to a virus isolate ofavian pnuemovirus (APV) also known as turkey rhinotracheitis virus(TRTV), the etiological agent of avian rhinotracheitis. It isparticularly useful to use an AVP-C virus isolate as outgroup in thephylogenetic tree analysis, it being the closest relative, albeit beingan essentially non-mammalian virus.

5.9.1 Bioinformatics Alignment of Sequences

Two or more amino acid sequences can be compared by BLAST (S. F.Altschul, et al., 1990, J. Mol. Biol. 215:403-410) to determine theirsequence homology and sequence identities to each other. Two or morenucleotide sequences can be compared by BLAST (S. F. Altschul, et al.,1990, J. Mol. Biol. 215:403-410) to determine their sequence homologyand sequence identities to each other. BLAST comparisons can beperformed using the Clustal W method (MACVECTOR™). In certain specificembodiments, the alignment of two or more sequences by a computerprogram can be followed by manual re-adjustment.

The determination of percent identity between two sequences can beaccomplished using a mathematical algorithm. A preferred, non-limitingexample of a mathematical algorithm utilized for the comparison of twosequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl.Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul, 1993,Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm isincorporated into the NBLAST and XBLAST programs of Altschul et al.,1990, J. Mol. Biol. 215:403-410. BLAST nucleotide comparisons can beperformed with the NBLAST program. BLAST amino acid sequence comparisonscan be performed with the XBLAST program. To obtain gapped alignmentsfor comparison purposes, Gapped BLAST can be utilized as described inAltschul et al., 1997, Nucleic Acids Res. 25:3389-3402. Alternatively,PSI-Blast can be used to perform an iterated search which detectsdistant relationships between molecules (Altschul et al., 1997, supra).When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the defaultparameters of the respective programs (e.g., XBLAST and NBLAST) can beused (see on the World Wide Web at ncbi.nlm.nih.gov). Another preferred,non-limiting example of a mathematical algorithm utilized for thecomparison of sequences is the algorithm of Myers and Miller, 1988,CABIOS 4:11-17. Such an algorithm is incorporated into the ALIGN program(version 2.0) which is part of the GCG sequence alignment softwarepackage. When utilizing the ALIGN program for comparing amino acidsequences, a PAM120 weight residue table can be used. The gap lengthpenalty can be set by the skilled artisan. The percent identity betweentwo sequences can be determined using techniques similar to thosedescribed above, with or without allowing gaps. In calculating percentidentity, typically only exact matches are counted.

5.9.2 Hybridization Conditions

A nucleic acid which is hybridizable to a nucleic acid of a mammalianMPV, or to its reverse complement, or to its complement can be used inthe methods of the invention to determine their sequence homology andidentities to each other. In certain embodiments, the nucleic acids arehybridized under conditions of high stringency. By way of example andnot limitation, procedures using such conditions of high stringency areas follows. Prehybridization of filters containing DNA is carried outfor 8 hours to overnight at 65° C. in buffer composed of 6×SSC, 50 mMTris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and500 μg/ml denatured salmon sperm DNA. Filters are hybridized for 48hours at 65° C. in prehybridization mixture containing 100 μg/mldenatured salmon sperm DNA and 5-20×10⁶ cpm of 32P-labeled probe.Washing of filters is done at 37° C. for 1 hour in a solution containing2×SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA. This is followed by awash in 0.1×SSC at 50° C. for 45 minutes before autoradiography. Otherconditions of high stringency which may be used are well known in theart. In other embodiments of the invention, hybridization is performedunder moderate of low stringency conditions, such conditions arewell-known to the skilled artisan (see e.g., Sambrook et al., 1989,Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.; see also, Ausubel et al.,eds., in the Current Protocols in Molecular Biology series of laboratorytechnique manuals, 1987-1997 Current Protocols,© 1994-1997 John Wileyand Sons, Inc.).

5.9.3 Phylogenetic Analysis

This invention relates to the inference of phylogenetic relationshipsbetween isolates of mammalian MPV. Many methods or approaches areavailable to analyze phylogenetic relationship; these include distance,maximum likelihood, and maximum parsimony methods (D. L. Swofford, et.al., Phylogenetic Inference. In Molecular Systematics. Eds. D. M.Hillis, C. Mortiz, and B. K. Mable, 1996, Sinauer Associates:Massachusetts, USA. pp. 407-514; J. Felsenstein, 1981, J. Mol. Evol.17:368-376). In addition, bootstrapping techniques are an effectivemeans of preparing and examining confidence intervals of resultantphylogenetic trees (J. Felsenstein, 1985, Evolution. 29:783-791). Anymethod or approach using nucleotide or peptide sequence information tocompare mammalian MPV isolates can be used to establish phylogeneticrelationships, including, but not limited to, distance, maximumlikelihood, and maximum parsimony methods or approaches. Any methodknown in the art can be used to analyze the quality of phylogeneticdata, including but not limited to bootstrapping. Alignment ofnucleotide or peptide sequence data for use in phylogenetic approaches,include but are not limited to, manual alignment, computer pairwisealignment, and computer multiple alignment. One skilled in the art wouldbe familiar with the preferable alignment method or phylogeneticapproach to be used based upon the information required and the timeallowed.

In one embodiment, a DNA maximum likelihood method is used to inferrelationships between hMPV isolates. In another embodiment,bootstrapping techniques are used to determine the certainty ofphylogenetic data created using one of the phylogenetic approaches. Inanother embodiment, jumbling techniques are applied to the phylogeneticapproach before the input of data in order to minimize the effect ofsequence order entry on the phylogenetic analyses. In one specificembodiment, a DNA maximum likelihood method is used with bootstrapping.In another specific embodiment, a DNA maximum likelihood method is usedwith bootstrapping and jumbling. In another more specific embodiment, aDNA maximum likelihood method is used with 50 bootstraps. In anotherspecific embodiment, a DNA maximum likelihood method is used with 50bootstraps and 3 jumbles. In another specific embodiment, a DNA maximumlikelihood method is used with 100 bootstraps and 3 jumbles.

In one embodiment, nucleic acid or peptide sequence information from anisolate of hMPV is compared or aligned with sequences of other hMPVisolates. The amino acid sequence can be the amino acid sequence of theL protein, the M protein, the N protein, the P protein, or the Fprotein. In another embodiment, nucleic acid or peptide sequenceinformation from an hMPV isolate or a number of hMPV isolates iscompared or aligned with sequences of other viruses. In anotherembodiment, phylogenetic approaches are applied to sequence alignmentdata so that phylogenetic relationships can be inferred and/orphylogenetic trees constructed. Any method or approach that usesnucleotide or peptide sequence information to compare hMPV isolates canbe used to infer the phylogenetic relationships, including, but notlimited to, distance, maximum likelihood, and maximum parsimony methodsor approaches.

Other methods for the phylogenetic analysis are disclosed inInternational Patent Application PCT/NL02/00040, published as WO02/057302, which is incorporated in its entirety herein. In particular,PCT/NL02/00040 discloses nucleic acid sequences that are suitable forphylogenetic analysis at page 12, line 27 to page 19, line 29, which isincorporated herein by reference.

For the phylogenetic analyses it is most useful to obtain the nucleicacid sequence of a non-MPV as outgroup with which the virus is to becompared, a very useful outgroup isolate can be obtained from avianpneumovirus serotype C (APV-C), see, e.g., FIG. 16.

Many methods and programs are known in the art and can be used in theinference of phylogenetic relationships, including, but not limited toBioEdit, ClustalW, TreeView, and NJPlot. Methods that would be used toalign sequences and to generate phylogenetic trees or relationshipswould require the input of sequence information to be compared. Manymethods or formats are known in the art and can be used to inputsequence information, including, but not limited to, FASTA, NBRF,EMBL/SWISS, GDE protein, GDE nucleotide, CLUSTAL, and GCG/MSF. Methodsthat would be used to align sequences and to generate phylogenetic treesor relationships would require the output of results. Many methods orformats can be used in the output of information or results, including,but not limited to, CLUSTAL, NBRF/PIR, MSF, PHYLIP, and GDE. In oneembodiment, ClustalW is used in conjunction with DNA maximum likelihoodmethods with 100 bootstraps and 3 jumbles in order to generatephylogenetic relationships.

5.10 Generation of Antibodies

The invention also relates to the generation of antibodies against aprotein encoded by a mammalian MPV. In particular, the invention relatesto the generation of antibodies against all MPV antigens, including theF protein, N protein, M2-1 protein, M2-2 protein, G protein, or Pprotein of a mammalian MPV. According to the invention, any proteinencoded by a mammalian MPV, derivatives, analogs or fragments thereof,may be used as an immunogen to generate antibodies whichimmunospecifically bind such an immunogen. Antibodies of the inventioninclude, but are not limited to, polyclonal, monoclonal, multispecific,human, humanized or chimeric antibodies, single chain antibodies, Fabfragments, F(ab′) fragments, fragments produced by a Fab expressionlibrary, anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Idantibodies to antibodies of the invention), and epitope-bindingfragments. The term “antibody,” as used herein, refers to immunoglobulinmolecules and immunologically active portions of immunoglobulinmolecules, i.e., molecules that contain an antigen binding site thatimmunospecifically binds an antigen. The immunoglobulin molecules of theinvention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY),class (e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁ and IgA₂) or subclass ofimmunoglobulin molecule. Examples of immunologically active portions ofimmunoglobulin molecules include F(ab) and F(ab′)₂ fragments which canbe generated by treating the antibody with an enzyme such as pepsin orpapain. In a specific embodiment, antibodies to a protein encoded byhuman MPV are produced. In another embodiment, antibodies to a domain aprotein encoded by human MPV are produced.

Various procedures known in the art may be used for the production ofpolyclonal antibodies against a protein encoded by a mammalian MPV,derivatives, analogs or fragments thereof. For the production ofantibody, various host animals can be immunized by injection with thenative protein, or a synthetic version, or derivative (e.g., fragment)thereof, including but not limited to rabbits, mice, rats, etc. Variousadjuvants may be used to increase the immunological response, dependingon the host species, and including but not limited to Freund's (completeand incomplete), mineral gels such as aluminum hydroxide, surface activesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,oil emulsions, keyhole limpet hemocyanins, dinitrophenol, andpotentially useful human adjuvants such as BCG (bacille Calmette-Guerin)and corynebacterium parvum.

For preparation of monoclonal antibodies directed toward a proteinencoded by a mammalian MPV, derivatives, analogs or fragments thereof,any technique which provides for the production of antibody molecules bycontinuous cell lines in culture may be used. For example, the hybridomatechnique originally developed by Kohler and Milstein (1975, Nature256:495-497), as well as the trioma technique, the human B-cellhybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), andthe EBV-hybridoma technique to produce human monoclonal antibodies (Coleet al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc., pp. 77-96). In an additional embodiment of the invention,monoclonal antibodies can be produced in germ-free animals utilizingrecent technology (PCT/US90/02545). According to the invention, humanantibodies may be used and can be obtained by using human hybridomas(Cote et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:2026-2030) or bytransforming human B cells with EBV virus in vitro (Cole et al., 1985,in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, pp. 77-96).In fact, according to the invention, techniques developed for theproduction of “chimeric antibodies” (Morrison et al., 1984, Proc. Natl.Acad. Sci. U.S.A. 81:6851-6855; Neuberger et al., 1984, Nature312:604-608; Takeda et al., 1985, Nature 314:452-454) by splicing thegenes from a mouse antibody molecule specific for a protein encoded by amammalian MPV, derivatives, analogs or fragments thereof together withgenes from a human antibody molecule of appropriate biological activitycan be used; such antibodies are within the scope of this invention.

According to the invention, techniques described for the production ofsingle chain antibodies (U.S. Pat. No. 4,946,778) can be adapted toproduce specific single chain antibodies. An additional embodiment ofthe invention utilizes the techniques described for the construction ofFab expression libraries (Huse et al., 1989, Science 246:1275-1281) toallow rapid and easy identification of monoclonal Fab fragments with thedesired specificity for a protein encoded by a mammalian MPV,derivatives, analogs or fragments thereof.

Antibody fragments which contain the idiotype of the molecule can begenerated by known techniques. For example, such fragments include butare not limited to: the F(ab′)₂ fragment which can be produced by pepsindigestion of the antibody molecule; the Fab′ fragments which can begenerated by reducing the disulfide bridges of the F(ab′)₂ fragment, theFab fragments which can be generated by treating the antibody moleculewith papain and a reducing agent, and Fv fragments.

In the production of antibodies, screening for the desired antibody canbe accomplished by techniques known in the art, e.g. ELISA(enzyme-linked immunosorbent assay). For example, to select antibodieswhich recognize a specific domain of a protein encoded by a mammalianMPV, one may assay generated hybridomas for a product which binds to afragment of a protein encoded by a mammalian MPV containing such domain.

The antibodies provided by the present invention can be used fordetecting MPV and for therapeutic methods for the treatment ofinfections with MPV.

The specificity and binding affinities of the antibodies generated bythe methods of the invention can be tested by any technique known to theskilled artisan. In certain embodiments, the specificity and bindingaffinities of the antibodies generated by the methods of the inventioncan be tested as described in sections 5.8.5, 5.8.6, 5.8.7, 5.8.8 or5.8.9.

5.11 Screening Assays to Identify Antiviral Agents

The invention provides methods for the identification of a compound thatinhibits the ability of a mammalian MPV to infect a host or a host cell.In certain embodiments, the invention provides methods for theidentification of a compound that reduces the ability of a mammalian MPVto replicate in a host or a host cell. Any technique well-known to theskilled artisan can be used to screen for a compound that would abolishor reduce the ability of a mammalian MPV to infect a host and/or toreplicate in a host or a host cell. In a specific embodiment, themammalian MPV is a human MPV.

In certain embodiments, the invention provides methods for theidentification of a compound that inhibits the ability of a mammalianMPV to replicate in a mammal or a mammalian cell. More specifically, theinvention provides methods for the identification of a compound thatinhibits the ability of a mammalian MPV to infect a mammal or amammalian cell. In certain embodiments, the invention provides methodsfor the identification of a compound that inhibits the ability of amammalian MPV to replicate in a mammalian cell. In a specificembodiment, the mammalian cell is a human cell. For a detaileddescription of assays that can be used to determine virus titer seesection 5.7.

In certain embodiments, a cell is contacted with a test compound andinfected with a mammalian MPV. In certain embodiments, a control cultureis infected with a mammalian virus in the absence of a test compound.The cell can be contacted with a test compound before, concurrentlywith, or subsequent to the infection with the mammalian MPV. In aspecific embodiment, the cell is a mammalian cell. In an even morespecific embodiment, the cell is a human cell. In certain embodiments,the cell is incubated with the test compound for at least 1 minute, atleast 5 minutes at least 15 minutes, at least 30 minutes, at least 1hour, at least 2 hours, at least 5 hours, at least 12 hours, or at least1 day. The titer of the virus can be measured at any time during theassay. In certain embodiments, a time course of viral growth in theculture is determined. If the viral growth is inhibited or reduced inthe presence of the test compound, the test compound is identified asbeing effective in inhibiting or reducing the growth or infection of amammalian MPV. In a specific embodiment, the compound that inhibits orreduces the growth of a mammalian MPV is tested for its ability toinhibit or reduce the growth rate of other viruses to test itsspecificity for mammalian MPV.

In certain embodiments, a test compound is administered to a modelanimal and the model animal is infected with a mammalian MPV. In certainembodiments, a control model animal is infected with a mammalian virusin without the administration of a test compound. The test compound canbe administered before, concurrently with, or subsequent to theinfection with the mammalian MPV. In a specific embodiment, the modelanimal is a mammal. In an even more specific embodiment, the modelanimal can be, but is not limited to, a cotton rat, a mouse, or amonkey. The titer of the virus in the model animal can be measured atany time during the assay. In certain embodiments, a time course ofviral growth in the culture is determined. If the viral growth isinhibited or reduced in the presence of the test compound, the testcompound is identified as being effective in inhibiting or reducing thegrowth or infection of a mammalian MPV. In a specific embodiment, thecompound that inhibits or reduces the growth of a mammalian MPV in themodel animal is tested for its ability to inhibit or reduce the growthrate of other viruses to test its specificity for mammalian MPV.

5.12 Formulations of Vaccines, Antibodies and Antivirals

In a preferred embodiment, the invention provides a proteinaceousmolecule or metapneumovirus-specific viral protein or functionalfragment thereof encoded by a nucleic acid according to the invention.Useful proteinaceous molecules are for example derived from any of thegenes or genomic fragments derivable from a virus according to theinvention. Such molecules, or antigenic fragments thereof, as providedherein, are for example useful in diagnostic methods or kits and inpharmaceutical compositions such as sub-unit vaccines. Particularlyuseful are the F, SH and/or G protein or antigenic fragments thereof forinclusion as antigen or subunit immunogen, but inactivated whole viruscan also be used. Particularly useful are also those proteinaceoussubstances that are encoded by recombinant nucleic acid fragments thatare identified for phylogenetic analyses, of course preferred are thosethat are within the preferred bounds and metes of ORFs useful inphylogenetic analyses, in particular for eliciting MPV specific antibodyor T cell responses, whether in vivo (e.g. for protective purposes orfor providing diagnostic antibodies) or in vitro (e.g. by phage displaytechnology or another technique useful for generating syntheticantibodies).

Also provided herein are antibodies, be it natural polyclonal ormonoclonal, or synthetic (e.g. (phage) library-derived bindingmolecules) antibodies that specifically react with an antigen comprisinga proteinaceous molecule or MPV-specific functional fragment thereofaccording to the invention. Such antibodies are useful in a method foridentifying a viral isolate as an MPV comprising reacting the viralisolate or a component thereof with an antibody as provided herein. Thiscan for example be achieved by using purified or non-purified MPV orparts thereof (proteins, peptides) using ELISA, RIA, FACS or differentformats of antigen detection assays (Current Protocols in Immunology).Alternatively, infected cells or cell cultures may be used to identifyviral antigens using classical immunofluorescence or immunohistochemicaltechniques.

A pharmaceutical composition comprising a virus, a nucleic acid, aproteinaceous molecule or fragment thereof, an antigen and/or anantibody according to the invention can for example be used in a methodfor the treatment or prevention of a MPV infection and/or a respiratoryillness comprising providing an individual with a pharmaceuticalcomposition according to the invention. This is most useful when theindividual comprises a human, specifically when the human is below 5years of age, since such infants and young children are most likely tobe infected by a human MPV as provided herein. Generally, in the acutephase patients will suffer from upper respiratory symptoms predisposingfor other respiratory and other diseases. Also lower respiratoryillnesses may occur, predisposing for more and other serious conditions.The compositions of the invention can be used for the treatment ofimmuno-compromised individuals including cancer patients, transplantrecipients and the elderly.

The invention also provides methods to obtain an antiviral agent usefulin the treatment of respiratory tract illness comprising establishing acell culture or experimental animal comprising a virus according to theinvention, treating the culture or animal with an candidate antiviralagent, and determining the effect of the agent on the virus or itsinfection of the culture or animal. An example of such an antiviralagent comprises a MPV-neutralizing antibody, or functional componentthereof, as provided herein, but antiviral agents of other nature areobtained as well. The invention also provides use of an antiviral agentaccording to the invention for the preparation of a pharmaceuticalcomposition, in particular for the preparation of a pharmaceuticalcomposition for the treatment of respiratory tract illness, specificallywhen caused by an MPV infection or related disease, and provides apharmaceutical composition comprising an antiviral agent according tothe invention, useful in a method for the treatment or prevention of anMPV infection or respiratory illness, the method comprising providing anindividual with such a pharmaceutical composition.

In certain embodiments of the invention, the vaccine of the inventioncomprises mammalian metapneumovirus as defined herein. In certain, morespecific embodiments, the mammalian metapneumovirus is a humanmetapneumovirus. In a preferred embodiment, the mammalianmetapneumovirus to be used in a vaccine formulation has an attenuatedphenotype. For methods to achieve an attenuated phenotype, see section5.6.

The invention provides vaccine formulations for the prevention andtreatment of infections with PIV, RSV, APV, and/or hMPV. In certainembodiments, the vaccine of the invention comprises recombinant andchimeric viruses of the invention. In certain embodiments, the virus isattenuated.

In a specific embodiment, the vaccine comprises APV and the vaccine isused for the prevention and treatment for hMPV infections in humans.Without being bound by theory, because of the high degree of homology ofthe F protein of APV with the F protein of hMPV, infection with APV willresult in the production of antibodies in the host that will cross-reactwith hMPV and protect the host from infection with hMPV and relateddiseases.

In another specific embodiment, the vaccine comprises hMPV and thevaccine is used for the prevention and treatment for APV infection inbirds, such as, but not limited to, in turkeys. Without being bound bytheory, because of the high degree of homology of the F protein of APVwith the F protein of hMPV, infection with hMPV will result in theproduction of antibodies in the host that will cross-react with APV andprotect the host from infection with APV and related diseases.

In a specific embodiment, the invention encompasses the use ofrecombinant and chimeric APV/hMPV viruses which have been modified invaccine formulations to confer protection against APV and/or hMPV. Incertain embodiments, APV/hMPV is used in a vaccine to be administered tobirds, to protect the birds from infection with APV. Without being boundby theory, the replacement of the APV gene or nucleotide sequence with ahMPV gene or nucleotide sequence results in an attenuated phenotype thatallows the use of the chimeric virus as a vaccine. In other embodimentsthe APV/hMPV chimeric virus is administered to humans. Without beingbound by theory the APV viral vector provides the attenuated phenotypein humans and the expression of the hMPV sequence elicits a hMPVspecific immune response.

In a specific embodiment, the invention encompasses the use ofrecombinant and chimeric hMPV/APV viruses which have been modified invaccine formulations to confer protection against APV and/or hMPV. Incertain embodiments, hMPV/APV is used in a vaccine to be administered tohumans, to protect the human from infection with hMPV. Without beingbound by theory, the replacement of the hMPV gene or nucleotide sequencewith a APV gene or nucleotide sequence results in an attenuatedphenotype that allows the use of the chimeric virus as a vaccine. Inother embodiments the hMPV/APV chimeric virus is administered to birds.Without being bound by theory the hMPV backbone provides the attenuatedphenotype in birds and the expression of the APV sequence elicits an APVspecific immune response.

In certain preferred embodiments, the vaccine formulation of theinvention is used to protect against infections by a metapneumovirus andrelated diseases. More specifically, the vaccine formulation of theinvention is used to protect against infections by a humanmetapneumovirus and/or an avian pneumovirus and related diseases. Incertain embodiments, the vaccine formulation of the invention is used toprotect against infections by (a) a human metapneumovirus and arespiratory syncytial virus; and/or (b) an avian pneumovirus and arespiratory syncytial virus.

In certain embodiments, the vaccine formulation of the invention is usedto protect against infections by (a) a human metapneumovirus and a humanparainfluenza virus; and/or (b) an avian pneumovirus and a humanparainfluenza virus, and related diseases.

In certain embodiments, the vaccine formulation of the invention is usedto protect against infections by (a) a human metapneumovirus, arespiratory syncytial virus, and a human parainfluenza virus; and/or (b)an avian pneumovirus, a respiratory syncytial virus, and a humanparainfluenza virus, and related diseases.

In certain embodiments, the vaccine formulation of the invention is usedto protect against infections by a human metapneumovirus, a respiratorysyncytial virus, and a human parainfluenza virus and related diseases.In certain other embodiments, the vaccine formulation of the inventionis used to protect against infections by an avian pneumovirus, arespiratory syncytial virus, and a human parainfluenza virus and relateddiseases.

Due to the high degree of homology among the F proteins of differentviral species, for exemplary amino acid sequence comparisons see FIG. 9,the vaccine formulations of the invention can be used for protectionfrom viruses different from the one from which the heterologousnucleotide sequence encoding the F protein was derived. In a specificexemplary embodiment, a vaccine formulation contains a virus comprisinga heterologous nucleotide sequence derived from an avian pneumovirustype A, and the vaccine formulation is used to protect from infection byavian pneumovirus type A and avian pneumovirus type B.

The invention encompasses vaccine formulations to be administered tohumans and animals which are useful to protect against APV, includingAPV-C and APV-D, kMPV, PIV, influenza, RSV, Sendai virus, mumps,laryngotracheitis virus, simianvirus 5, human papillomavirus, measles,mumps, as well as other viruses and pathogens and related diseases. Theinvention further encompasses vaccine formulations to be administered tohumans and animals which are useful to protect against humanmetapneumovirus infections and avian pneumovirus infections and relateddiseases.

In one embodiment, the invention encompasses vaccine formulations whichare useful against domestic animal disease causing agents includingrabies virus, feline leukemia virus (FLV) and canine distemper virus. Inyet another embodiment, the invention encompasses vaccine formulationswhich are useful to protect livestock against vesicular stomatitisvirus, rabies virus, rinderpest virus, swinepox virus, and further, toprotect wild animals against rabies virus.

Attenuated viruses generated by the reverse genetics approach can beused in the vaccine and pharmaceutical formulations described herein.Reverse genetics techniques can also be used to engineer additionalmutations to other viral genes important for vaccine production—i.e.,the epitopes of useful vaccine strain variants can be engineered intothe attenuated virus. Alternatively, completely foreign epitopes,including antigens derived from other viral or non-viral pathogens canbe engineered into the attenuated strain. For example, antigens ofnon-related viruses such as HIV (gp160, gp120, gp41) parasite antigens(e.g., malaria), bacterial or fungal antigens or tumor antigens can beengineered into the attenuated strain. Alternatively, epitopes whichalter the tropism of the virus in vivo can be engineered into thechimeric attenuated viruses of the invention.

Virtually any heterologous gene sequence may be constructed into thechimeric viruses of the invention for use in vaccines. Preferablymoieties and peptides that act as biological response modifiers.Preferably, epitopes that induce a protective immune response to any ofa variety of pathogens, or antigens that bind neutralizing antibodiesmay be expressed by or as part of the chimeric viruses. For example,heterologous gene sequences that can be constructed into the chimericviruses of the invention include, but are not limited to influenza andparainfluenza hemagglutinin neuraminidase and fusion glycoproteins suchas the HN and F genes of human PIV3. In yet another embodiment,heterologous gene sequences that can be engineered into the chimericviruses include those that encode proteins with immuno-modulatingactivities. Examples of immuno-modulating proteins include, but are notlimited to, cytokines, interferon type 1, gamma interferon, colonystimulating factors, interleukin-2, -4, -5, -6, -12, and antagonists ofthese agents.

In addition, heterologous gene sequences that can be constructed intothe chimeric viruses of the invention for use in vaccines include butare not limited to sequences derived from a human immunodeficiency virus(HIV), preferably type 1 or type 2. In a preferred embodiment, animmunogenic HIV-derived peptide which may be the source of an antigenmay be constructed into a chimeric PIV that may then be used to elicit avertebrate immune response. Such HIV-derived peptides may include, butare not limited to sequences derived from the env gene (i.e., sequencesencoding all or part of gp160, gp120, and/or gp41), the pot gene (i.e.,sequences encoding all or part of reverse transcriptase, endonuclease,protease, and/or integrase), the gag gene (i.e., sequences encoding allor part of p7, p6, p55, p17/18, p24/25), tat, rev, nef, vif, vpu, vpr,and/or vpx.

Other heterologous sequences may be derived from hepatitis B virussurface antigen (HBsAg); hepatitis A or C virus surface antigens, theglycoproteins of Epstein Barr virus; the glycoproteins of humanpapillomavirus; the glycoproteins of respiratory syncytial virus,parainfluenza virus, Sendai virus, simianvirus 5 or mumps virus; theglycoproteins of influenza virus; the glycoproteins of herpes viruses;VP1 of poliovirus; antigenic determinants of non-viral pathogens such asbacteria and parasites, to name but a few. In another embodiment, all orportions of immunoglobulin genes may be expressed. For example, variableregions of anti-idiotypic immunoglobulins that mimic such epitopes maybe constructed into the chimeric viruses of the invention.

Other heterologous sequences may be derived from tumor antigens, and theresulting chimeric viruses be used to generate an immune responseagainst the tumor cells leading to tumor regression in vivo. Thesevaccines may be used in combination with other therapeutic regimens,including but not limited to chemotherapy, radiation therapy, surgery,bone marrow transplantation, etc. for the treatment of tumors. Inaccordance with the present invention, recombinant viruses may beengineered to express tumor-associated antigens (TAAs), including butnot limited to, human tumor antigens recognized by T cells (Robbins andKawakami, 1996, Curr. Opin. Immunol. 8:628-636, incorporated herein byreference in its entirety), melanocyte lineage proteins, includinggp100, MART-1/MelanA, TRP-1 (gp75), tyrosinase; Tumor-specific widelyshared antigens, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-1,N-acetylglucosaminyltransferase-V, p15; Tumor-specific mutated antigens,β-catenin, MUM-1, CDK4; Nonmelanoma antigens for breast, ovarian,cervical and pancreatic carcinoma, HER-2/neu, human papillomavirus-E6,-E7, MUC-1.

In even other embodiments, a heterologous nucleotide sequence is derivedfrom a metapneumovirus, such as human metapneumovirus and/or avianpneumovirus. In even other embodiments, the virus of the inventioncontains two different heterologous nucleotide sequences wherein one isderived from a metapneumovirus, such as human metapneumovirus and/oravian pneumovirus, and the other one is derived from a respiratorysyncytial virus. The heterologous nucleotide sequence encodes a Fprotein or a G protein of the respective virus. In a specificembodiment, a heterologous nucleotide sequences encodes a chimeric Fprotein, wherein the chimeric F protein contains the ectodomain of a Fprotein of a metapneumovirus and the transmembrane domain as well as theluminal domain of a F protein of a parainfluenza virus.

Either a live recombinant viral vaccine or an inactivated recombinantviral vaccine can be formulated. A live vaccine may be preferred becausemultiplication in the host leads to a prolonged stimulus of similar kindand magnitude to that occurring in natural infections, and therefore,confers substantial, long-lasting immunity. Production of such liverecombinant virus vaccine formulations may be accomplished usingconventional methods involving propagation of the virus in cell cultureor in the allantois of the chick embryo followed by purification.

In a specific embodiment, the recombinant virus is non-pathogenic to thesubject to which it is administered. In this regard, the use ofgenetically engineered viruses for vaccine purposes may desire thepresence of attenuation characteristics in these strains. Theintroduction of appropriate mutations (e.g., deletions) into thetemplates used for transfection may provide the novel viruses withattenuation characteristics. For example, specific missense mutationswhich are associated with temperature sensitivity or cold adaption canbe made into deletion mutations. These mutations should be more stablethan the point mutations associated with cold or temperature sensitivemutants and reversion frequencies should be extremely low.

Alternatively, chimeric viruses with “suicide” characteristics may beconstructed. Such viruses would go through only one or a few rounds ofreplication within the host. When used as a vaccine, the recombinantvirus would go through limited replication cycle(s) and induce asufficient level of immune response but it would not go further in thehuman host and cause disease. Recombinant viruses lacking one or more ofthe genes of wild type APV and hMPV, respectively, or possessing mutatedgenes as compared to the wild type strains would not be able to undergosuccessive rounds of replication. Defective viruses can be produced incell lines which permanently express such a gene(s). Viruses lacking anessential gene(s) will be replicated in these cell lines but whenadministered to the human host will not be able to complete a round ofreplication. Such preparations may transcribe and translate—in thisabortive cycle—a sufficient number of genes to induce an immuneresponse. Alternatively, larger quantities of the strains could beadministered, so that these preparations serve as inactivated (killed)virus vaccines. For inactivated vaccines, it is preferred that theheterologous gene product be expressed as a viral component, so that thegene product is associated with the virion. The advantage of suchpreparations is that they contain native proteins and do not undergoinactivation by treatment with formalin or other agents used in themanufacturing of killed virus vaccines. Alternatively, recombinant virusof the invention made from cDNA may be highly attenuated so that itreplicates for only a few rounds.

In certain embodiments, the vaccine of the invention comprises anattenuated mammalian MPV. Without being bound by theory, the attenuatedvirus can be effective as a vaccine even if the attenuated virus isincapable of causing a cell to generate new infectious viral particlesbecause the viral proteins are inserted in the cytoplasmic membrane ofthe host thus stimulating an immune response.

In another embodiment of this aspect of the invention, inactivatedvaccine formulations may be prepared using conventional techniques to“kill” the chimeric viruses. Inactivated vaccines are “dead” in thesense that their infectivity has been destroyed. Ideally, theinfectivity of the virus is destroyed without affecting itsimmunogenicity. In order to prepare inactivated vaccines, the chimericvirus may be grown in cell culture or in the allantois of the chickembryo, purified by zonal ultracentrifugation, inactivated byformaldehyde or β-propiolactone, and pooled. The resulting vaccine isusually inoculated intramuscularly.

Inactivated viruses may be formulated with a suitable adjuvant in orderto enhance the immunological response. Such adjuvants may include butare not limited to mineral gels, e.g., aluminum hydroxide; surfaceactive substances such as lysolecithin, pluronic polyols, polyanions;peptides; oil emulsions; and potentially useful human adjuvants such asBCG, Corynebacterium parvum, ISCOMS and virosomes.

Many methods may be used to introduce the vaccine formulations describedabove, these include but are not limited to oral, intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, percutaneous,and intranasal and inhalation routes. It may be preferable to introducethe chimeric virus vaccine formulation via the natural route ofinfection of the pathogen for which the vaccine is designed.

In certain embodiments, the invention relates to immunogeniccompositions. The immunogenic compositions comprise a mammalian MPV. Ina specific embodiment, the immunogenic composition comprises a humanMPV. In certain embodiments, the immunogenic composition comprises anattenuated mammalian MPV or an attenuated human MPV. In certainembodiments, the immunogenic composition further comprises apharmaceutically acceptable carrier.

5.13 Dosage Regimens, Administration and Formulations

The present invention provides vaccines and immunogenic preparationscomprising MPV and APV, including attenuated forms of the virus,recombinant forms of MPV and APV, and chimeric MPV and APV expressingone or more heterologous or non-native antigenic sequences. The vaccinesor immunogenic preparations of the invention encompass single ormultivalent vaccines, including bivalent and trivalent vaccines. Thevaccines or immunogenic formulations of the invention are useful inproviding protections against various viral infections. Particularly,the vaccines or immunogenic formulations of the invention provideprotection against respiratory tract infections in a host.

A recombinant virus and/or a vaccine or immunogenic formulation of theinvention can be administered alone or in combination with othervaccines. Preferably, a vaccine or immunogenic formulation of theinvention is administered in combination with other vaccines orimmunogenic formulations that provide protection against respiratorytract diseases, such as but not limited to, respiratory syncytial virusvaccines, influenza vaccines, measles vaccines, mumps vaccines, rubellavaccines, pneumococcal vaccines, rickettsia vaccines, staphylococcusvaccines, whooping cough vaccines or vaccines against respiratory tractcancers. In a preferred embodiment, the virus and/or vaccine of theinvention is administered concurrently with pediatric vaccinesrecommended at the corresponding ages. For example, at two, four or sixmonths of age, the virus and/or vaccine of the invention can beadministered concurrently with DtaP (IM), Hib (IM), Polio (IPV or OPV)and Hepatitis B (IM). At twelve or fifteen months of age, the virusand/or vaccine of the invention can be administered concurrently withHib (TM), Polio (IPV or OPV), MMRII® (SubQ); VARIVAX® (SubQ), andhepatitis B (IM). The vaccines that can be used with the methods ofinvention are reviewed in various publications, e.g., The Jordan Report2000, Division of Microbiology and Infectious Diseases, NationalInstitute of Allergy and Infectious Diseases, National Institutes ofHealth, United States, the content of which is incorporated herein byreference in its entirety.

A vaccine or immunogenic formulation of the invention may beadministered to a subject per se or in the form of a pharmaceutical ortherapeutic composition. Pharmaceutical compositions comprising anadjuvant and an immunogenic antigen of the invention (e.g., a virus, achimeric virus, a mutated virus) may be manufactured by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping or lyophilizing processes.Pharmaceutical compositions may be formulated in conventional mannerusing one or more physiologically acceptable carriers, diluents,excipients or auxiliaries which facilitate processing of the immunogenicantigen of the invention into preparations which can be usedpharmaceutically. Proper formulation is, amongst others, dependent uponthe route of administration chosen.

When a vaccine or immunogenic composition of the invention comprisesadjuvants or is administered together with one or more adjuvants, theadjuvants that can be used include, but are not limited to, mineral saltadjuvants or mineral salt gel adjuvants, particulate adjuvants,microparticulate adjuvants, mucosal adjuvants, and immunostimulatoryadjuvants. Examples of adjuvants include, but are not limited to,aluminum hydroxide, aluminum phosphate gel, Freund's Complete Adjuvant,Freund's Incomplete Adjuvant, squalene or squalene oil-in-water adjuvantformulations, biodegradable and biocompatible polyesters, polymerizedliposomes, triterpenoid glycosides or saponins (e.g., QuilA and QS-21,also sold under the trademark STIMULON, ISCOPREP),N-acetyl-muramyl-L-threonyl-D-isoglutamine (Threonyl-MDP, sold under thetrademark TERMURTIDE), LPS, monophosphoryl Lipid A (3D-MLA sold underthe trademark MPL).

The subject to which the vaccine or an immunogenic composition of theinvention is administered is preferably a mammal, most preferably ahuman, but can also be a non-human animal, including but not limited to,primates, cows, horses, sheep, pigs, fowl (e.g., chickens, turkeys),goats, cats, dogs, hamsters, mice and rodents.

Many methods may be used to introduce the vaccine or the immunogeniccomposition of the invention, including but not limited to, oral,intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous,percutaneous, intranasal and inhalation routes, and via scarification(scratching through the top layers of skin, e.g., using a bifurcatedneedle).

For topical administration, the vaccine or immunogenic preparations ofthe invention may be formulated as solutions, gels, ointments, creams,suspensions, etc. as are well-known in the art.

For administration intranasally or by inhalation, the preparation foruse according to the present invention can be conveniently delivered inthe form of an aerosol spray presentation from pressurized packs or anebulizer, with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof, e.g., gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch.

For injection, the vaccine or immunogenic preparations may be formulatedin aqueous solutions, preferably in physiologically compatible bufferssuch as Hanks's solution, Ringer's solution, or physiological salinebuffer. The solution may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the proteins may bein powder form for constitution with a suitable vehicle, e.g., sterilepyrogen-free water, before use.

Determination of an effective amount of the vaccine or immunogenicformulation for administration is well within the capabilities of thoseskilled in the art, especially in light of the detailed disclosureprovided herein.

An effective dose can be estimated initially from in vitro assays. Forexample, a dose can be formulated in animal models to achieve aninduction of an immunity response using techniques that are well knownin the art. One having ordinary skill in the art could readily optimizeadministration to all animal species based on results described herein.Dosage amount and interval may be adjusted individually. For example,when used as an immunogenic composition, a suitable dose is an amount ofthe composition that when administered as described above, is capable ofeliciting an antibody response. When used as a vaccine, the vaccine orimmunogenic formulations of the invention may be administered in about 1to 3 doses for a 1-36 week period. Preferably, 1 or 2 doses areadministered, at intervals of about 2 weeks to about 4 months, andbooster vaccinations may be given periodically thereafter. Alternateprotocols may be appropriate for individual animals. A suitable dose isan amount of the vaccine formulation that, when administered asdescribed above, is capable of raising an immunity response in animmunized animal sufficient to protect the animal from an infection forat least 4 to 12 months. In general, the amount of the antigen presentin a dose ranges from about 1 pg to about 100 mg per kg of host,typically from about 10 pg to about 1 mg, and preferably from about 100pg to about 1 μg. Suitable dose range will vary with the route ofinjection and the size of the patient, but will typically range fromabout 0.1 mL to about 5 mL.

In a specific embodiment, the viruses and/or vaccines of the inventionare administered at a starting single dose of at least 10³ TCID₅₀, atleast 10⁴ TCID₅₀, at least 10⁵ TCID₅₀, at least 10⁶ TCID₅₀. In anotherspecific embodiment, the virus and/or vaccines of the invention areadministered at multiple doses. In a preferred embodiment, a primarydosing regimen at 2, 4, and 6 months of age and a booster dose at thebeginning of the second year of life are used. More preferably, eachdose of at least 10⁵ TCID₅₀, or at least 10⁶ TCID₅₀ is given in amultiple dosing regimen.

5.13.1 Challenge Studies

This assay is used to determine the ability of the recombinant virusesof the invention and of the vaccines of the invention to prevent lowerrespiratory tract viral infection in an animal model system, such as,but not limited to, cotton rats or hamsters. The recombinant virusand/or the vaccine can be administered by intravenous (W) route, byintramuscular (IM) route or by intranasal route (IN). The recombinantvirus and/or the vaccine can be administered by any technique well-knownto the skilled artisan. This assay is also used to correlate the serumconcentration of antibodies with a reduction in lung titer of the virusto which the antibodies bind.

On day 0, groups of animals, such as, but not limited to cotton rats(Sigmodon hispidis, average weight 100 g) cynomolgous macacques (averageweight 2.0 kg) are administered the recombinant or chimeric virus or thevaccine of interest or BSA by intramuscular injection, by intravenousinjection, or by intranasal route. Prior to, concurrently with, orsubsequent to administration of the recombinant virus or the vaccine ofthe invention, the animals are infected with wild type virus wherein thewild type virus is the virus against which the vaccine was generated. Incertain embodiments, the animals are infected with the wild type virusat least 1 day, at least 2 days, at least 3 days, at least 4 days, atleast 5 days, at least 6 days, 1 week or 1 or more months subsequent tothe administration of the recombinant virus and/or the vaccine of theinvention.

After the infection, cotton rats are sacrificed, and their lung tissueis harvested and pulmonary virus titers are determined by plaquetitration. Bovine serum albumin (BSA) 10 mg/kg is used as a negativecontrol. Antibody concentrations in the serum at the time of challengeare determined using a sandwich ELISA. Similarly, in macacques, virustiters in nasal and lung lavages can be measured.

5.13.2 Target Populations

In certain embodiments of the invention, the target population for thetherapeutic and diagnostic methods of the invention is defined by age.In certain embodiments, the target population for the therapeutic and/ordiagnostic methods of the invention is characterized by a disease ordisorder in addition to a respiratory tract infection.

In a specific embodiment, the target population encompasses youngchildren, below 2 years of age. In a more specific embodiment, thechildren below the age of 2 years do not suffer from illnesses otherthan respiratory tract infection.

In other embodiments, the target population encompasses patients above 5years of age. In a more specific embodiment, the patients above the ageof 5 years suffer from an additional disease or disorder includingcystic fibrosis, leukemia, and non-Hodgkin lymphoma, or recentlyreceived bone marrow or kidney transplantation.

In a specific embodiment of the invention, the target populationencompasses subjects in which the hMPV infection is associated withimmunosuppression of the hosts. In a specific embodiment, the subject isan immunocompromised individual.

In certain embodiments, the target population for the methods of theinvention encompasses the elderly.

In a specific embodiment, the subject to be treated or diagnosed withthe methods of the invention was infected with hMPV in the wintermonths.

5.13.3 Clinical Trials

Vaccines of the invention or fragments thereof tested in in vitro assaysand animal models may be further evaluated for safety, tolerance andpharmacokinetics in groups of normal healthy adult volunteers. Thevolunteers are administered intramuscularly, intravenously or by apulmonary delivery system a single dose of a recombinant virus of theinvention and/or a vaccine of the invention. Each volunteer is monitoredat least 24 hours prior to receiving the single dose of the recombinantvirus of the invention and/or a vaccine of the invention and eachvolunteer will be monitored for at least 48 hours after receiving thedose at a clinical site. Then volunteers are monitored as outpatients ondays 3, 7, 14, 21, 28, 35, 42, 49, and 56 post-dose.

Blood samples are collected via an indwelling catheter or directvenipuncture using 10 ml red-top Vacutainer tubes at the followingintervals: (1) prior to administering the dose of the recombinant virusof the invention and/or a vaccine of the invention; (2) during theadministration of the dose of the recombinant virus of the inventionand/or a vaccine of the invention; (3) 5 minutes, 10 minutes, 15minutes, 20 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12hours, 24 hours, and 48 hours after administering the dose of therecombinant virus of the invention and/or a vaccine of the invention;and (4) 3 days, 7 days 14 days, 21 days, 28 days, 35 days, 42 days, 49days, and 56 days after administering the dose of the recombinant virusof the invention and/or a vaccine of the invention. Samples are allowedto clot at room temperature and serum will be collected aftercentrifugation.

The amount of antibodies generated against the recombinant virus of theinvention and/or a vaccine of the invention in the samples from thepatients can be quantitated by ELISA. T-cell immunity (cytotoxic andhelper responses) in PBMC and lung and nasal lavages can also bemonitored.

The concentration of antibody levels in the serum of volunteers arecorrected by subtracting the predose serum level (background level) fromthe serum levels at each collection interval after administration of thedose of recombinant virus of the invention and/or a vaccine of theinvention. For each volunteer the pharmacokinetic parameters arecomputed according to the model-independent approach (Gibaldi et al.,eds., 1982, Pharmacokinetics, 2nd edition, Marcel Dekker, New York) fromthe corrected serum antibody or antibody fragment concentrations.

5.14 Methods for Detecting and Diagnosing Mammalian MPV

The invention provides means and methods for the diagnosis and/ordetection of MPV, the means and methods to be employed in the detectionof MPV, its components, and the products of its transcription,translation, expression, propagation, and metabolic processes. Morespecifically, this invention provides means and methods for thediagnosis of an MPV infection in animals and in humans, the means andmethods including but not limited to the detection of components of MPV,products of the life cycle of MPV, and products of a host's response toMPV exposure or infection.

In one embodiment, the invention provides means and methods for thediagnosis and detection of MPV, the means and methods including but notlimited to the detection of genomic material and other nucleic acidsthat are associated with or complimentary to MPV, the detection oftranscriptional and translational products of MPV, the products beingboth processed and unprocessed, and the detection of components of ahost response to MPV exposure or infection.

In one embodiment, the invention relates to the detection of MPV throughthe preparation and use of oligonucleotides that are complimentary tonucleic acid sequences and transcriptional products of nucleic acidsequences that are present within the genome of MPV. Furthermore, theinvention relates to the detection of nucleic acids, or sequencesthereof, that are present in the genome of MPV and its transcriptionproducts, using the oligonucleotides as primers for copying oramplification of specific regions of the MPV genome and its transcripts.The regions of the MPV genome and its transcripts that can be copied oramplified include but are not limited to complete and incompletestretches of one or more of the following: the N-gene, the P-gene, theM-gene, the F-gene, the M2-gene, the SH-gene, the G-gene, and theL-gene. In a specific embodiment, oligonucleotides are used as primersin conjunction with methods to copy or amplify the N-gene of MPV, ortranscripts thereof, for identification purposes. The methods includebut are not limited to RT-PCR assays, primer extension or run on assays,and other methods that employ the genetic material of MPV or transcriptsand compliments thereof as templates for the extension of nucleic acidsequences from the oligonucleotides.

In another embodiment, the invention relates to detection of MPV throughthe preparation and use of oligonucleotides that are complimentary tonucleic acid sequences and transcriptional products of nucleic acidsequences that are present within the genome of MPV. Furthermore, theinvention relates to the detection of nucleic acids, or sequencesthereof, that are present in or complimentary to the genome of MPV andits transcription products, using the oligonucleotide sequences asprobes for hybridization to and detection of specific regions within orcomplimentary to the MPV genome and its transcripts. The regions of theMPV genome and its transcripts that can be detected using hybridizationprobes include but are not limited to complete and incomplete stretchesof one or more of the following: the N-gene, the P-gene, the M-gene, theF-gene, the M2-gene, the SH-gene, the G-gene, and the L-gene. In aspecific embodiment, oligonucleotides are used as probes in conjunctionwith methods to detect, anneal, or hybridize to the N-gene of MPV, ortranscripts thereof, for identification purposes. The methods includebut are not limited to, Northern blots, Southern blots and other methodsthat employ the genetic material of MPV or transcripts and complimentsthereof as targets for the hybridization, annealing, or detection ofsequences or stretches of sequences within or complimentary to the MPVgenome.

A nucleic acid which is hybridizable to a nucleic acid of a mammalianMPV, or to its reverse complement, or to its complement can be used inthe methods of the invention to detect the presence of a mammalian MPV.In certain embodiments, the nucleic acids are hybridized underconditions of high stringency. By way of example and not limitation,procedures using such conditions of high stringency are as follows.Prehybridization of filters containing DNA is carried out for 8 hours toovernight at 65° C. in buffer composed of 6×SSC, 50 mM Tris-HCl (pH7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 μg/mldenatured salmon sperm DNA. Filters are hybridized for 48 hours at 65°C. in prehybridization mixture containing 100 μg/ml denatured salmonsperm DNA and 5-20×10⁶ cpm of 32P-labeled probe. Washing of filters isdone at 37° C. for 1 hour in a solution containing 2×SSC, 0.01% PVP,0.01% Ficoll, and 0.01% BSA. This is followed by a wash in 0.1×SSC at50° C. for 45 minutes before autoradiography. Other conditions of highstringency which may be used are well known in the art. In otherembodiments of the invention, hybridization is performed under moderateof low stringency conditions, such conditions are well-known to theskilled artisan (see e.g., Sambrook et al., 1989, Molecular Cloning, ALaboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.; see also, Ausubel et al., eds., in the CurrentProtocols in Molecular Biology series of laboratory technique manuals,1987-1997 Current Protocols,© 1994-1997 John Wiley and Sons, Inc.).

In another embodiment, the invention relates to the detection of an MPVinfection in an animal or human host through the preparation and use ofantibodies, e.g., monoclonal antibodies (MAbs), that are specific to andcan recognize peptides or nucleic acids that are characteristic of MPVor its gene products. The epitopes or antigenic determinants recognizedby the MAbs include but are not limited to proteinaceous and nucleicacid products that are synthesized during the life cycle and metabolicprocesses involved in MPV propagation. The proteinaceous or nucleic acidproducts that can be used as antigenic determinants for the generationof suitable antibodies include but are not limited to complete andincomplete transcription and expression products of one or more of thefollowing components of MPV: the N-gene, the P-gene, the M-gene, theF-gene, the M2-gene, the SH-gene, the G-gene, and the L-gene. In onespecific embodiment, MAbs raised against proteinaceous products of theG-gene or portions thereof are used in conjunction with other methods todetect or confirm the presence of the MPV expressed G peptide in abiological sample, e.g. body fluid. The methods include but are notlimited to ELISA, Radio-Immuno or Competition Assays,Immunoprecipitation and other methods that employ the transcribed orexpressed gene products of MPV as targets for detection by MAbs raisedagainst the targets or portions and relatives thereof.

In another embodiment, the invention relates to the detection of factorsthat are associated with and characteristic of a host's immunologicresponse to MPV exposure or infection. Upon exposure or infection byMPV, a host's immune system elicits a response to the exposure orinfection that involves the generation by the host of antibodiesdirected at eliminating or attenuating the effects and/or propagation ofvirus. This invention provides means and methods for the diagnosis ofMPV related disease through the detection of the antibodies that may beproduced as a result of MPV exposure to or infection of the host. Theepitopes recognized by the antibodies include but are not limited topeptides and their exposed surfaces that are accessible to a host immuneresponse and that can serve as antigenic determinants in the generationof an immune response by the host to the virus. Some of theproteinaceous and nuclear material used by a host immune response asepitopes for the generation of antibodies include but are not limited toproducts of one or more of the following components of MPV: the N-gene,the P-gene, the M-gene, the F-gene, the M2-gene, the SH-gene, theG-gene, and the L-gene. In one embodiment, antibodies to partially orcompletely accessible portions of the N-gene encoded peptides of MPV aredetected in a host sample. In a specific embodiment, proteinaceousproducts of the G-gene or portions thereof are used in conjunction withother methods to detect the presence of the host derived antibodies in abiological sample, e.g. body fluid. The methods include but are notlimited to ELISA, Radio-Immuno or Competition Assays, and other methodsthat employ the transcribed or expressed gene products of MPV as targetsfor detection by host antibodies that recognize the products and thatare found in biological samples.

This invention also provides means and methods for diagnostic assays ortest kits and for methods to detect agents of an MPV infection from avariety of sources including but not limited to biological samples,e.g., body fluids. In one embodiment, this invention relates to assays,kits, protocols, and procedures that are suitable for identifying an MPVnucleic acid or a compliment thereof. In another embodiment, thisinvention relates to assays, kits, protocols, and procedures that aresuitable for identifying an MPV expressed peptide or a portion thereof.In another embodiment, this invention relates to assays, kits,protocols, and procedures that are suitable for identifying componentsof a host immunologic response to MPV exposure or infection.

In addition to diagnostic confirmation of MPV infection of a host, thepresent invention also provides for means and methods to classifyisolates of MPV into distinct phylogenetic groups or subgroups. In oneembodiment, this feature can be used advantageously to distinguishbetween the different variant of MPV, variant A1, A2, B1 and B2, inorder to design more effective and subgroup specific therapies. Variantsof MPV can be differentiated on the basis of nucleotide or amino acidsequences of one or more of the following: the N-gene, the P-gene, theM-gene, the F-gene, the M2-gene, the SH-gene, the G-gene, and theL-gene. In a specific embodiment, MPV can be differentiated into aspecific subgroup using the nucleotide or amino acid sequence of the Ggene or glycoprotein and neutralization tests using monoclonalantibodies that also recognize the G glycoprotein.

In one embodiment, the diagnosis of an MPV infection in a human is madeusing any technique well known to one skilled in the art, e.g.,immunoassays. Immunoassays which can be used to analyze immunospecificbinding and cross-reactivity include, but are not limited to,competitive and non-competitive assay systems using techniques such aswestern blots, radioimmunoassays, ELISA (enzyme linked immunosorbentassay), sandwich immunoassays, immunoprecipitation assays, precipitinreactions, gel diffusion precipitation reactions, immunodiffusionassays, agglutination assays, complement-fixation assays, andfluorescent immunoassays, to name but a few. Such assays are routine andwell known in the art (see, e.g., Ausubel et al., eds, 1994, CurrentProtocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., NewYork, which is incorporated by reference herein in its entirety) andnon-limiting examples of immunoassays are described in section 5.8.

In one embodiment, the invention relates to the detection of an MPVinfection using oligonucleotides in conjunction with PCR or primerextension methods to copy or amplify regions of the MPV genome, theregions including but not limited to genes or parts of genes, e.g., theN, M, F, G, L, M, P, and M2 genes. In a specific embodiment,oligonucleotides are used in conjunction with RT-PCR methods. In afurther embodiment, the amplification products and/or genetic materialcan be probed with oligonucleotides that are complimentary to specificsequences that are either conserved between various hMPV strains or aredistinct amongst various hMPV strains. The latter set ofoligonucleotides would allow for identification of the specific strainof hMPV responsible for the infection of the host.

The invention provides methods for distinguishing between differentsubgroups and variants of hMPV that are capable of infecting a host. Inone specific embodiment, the hMPV that is responsible for a hostinfection is classified into a specific subgroup, e.g., subgroup A orsubgroup B. In another specific embodiment, the hMPV that is responsiblefor a host infection is classified as a specific variant of a subgroup,e.g., variant A1, A2, B1, or B2. In another embodiment, the inventionprovides means and methods for the classification of an hMPV that isresponsible for a host infection into a new subgroup and/or into a newvariant of a new or existing subgroup. The methods that are able todistinguish hMPV strains into subgroups and/or variant groups would beknown to one skilled in the art. In one embodiment, a polyclonalantibody is used to identify the etiological agent of an infection as astrain of hMPV, and a secondary antibody is used to distinguish thestrain as characteristic of a new or known subgroup and/or new or knownvariant of hMPV. In one embodiment, antibodies that are selective forhMPV are used in conjunction with immunoreactive assays, e.g. ELISA orRIA, to identify the presence of hMPV exposure or infection inbiological samples. In a further embodiment, secondary antibodies thatare selective for specific epitopes in the peptide sequence of hMPVproteins are used to further classify the etiological agents of theidentified hMPV infections into subgroups or variants. In one specificembodiment, an antibody raised against peptide epitopes that are sharedbetween all subgroups of hMPV is used to identify the etiological agentof an infection as an hMPV. In a further specific embodiment, antibodiesraised against peptide epitopes that are unique to the differentsubgroups and/or variants of hMPV are used to classify the hMPV that isresponsible for the host infection into a known or new subgroup and/orvariant. In one specific embodiment, the antibody that is capable ofdistinguishing between different subgroups and/or variants of hMPVrecognizes segments of hMPV peptides that are unique to the subgroup orvariant, the peptides including but not limited to those encoded by theN, M, F, G, L, M, P, and M2 genes. The peptides or segments of peptidesthat can be used to generate antibodies capable of distinguishingbetween different hMPV subgroups or variants can be selected usingdifferences in known peptide sequences of various hMPV proteins inconjunction with hydrophillicity plots to identify suitable peptidesegments that would be expected to be solvent exposed or accessible in adiagnostic assay. In one embodiment, the antibody that is capable ofdistinguishing between the different subgroups of hMPV recognizesdifferences in the F protein that are unique to different subgroups ofhMPV, e.g. the amino acids at positions 286, 296, 312, 348, and 404 ofthe full length F protein. In another specific embodiment, the antibodythat is capable of distinguishing between different subgroups and/orvariants of hMPV recognizes segments of the G protein of hMPV that areunique to specific subgroups or variants, e.g., the G peptide sequencecorresponding to amino acids 50 through 60 of SEQ ID:119 can be used todistinguish between subgroups A and B as well as between variants A1,A2, B1, and B2. In another embodiment of the invention, the nucleotidesequence of hMPV isolates are used to distinguish between differentsubgroups and/or different variants of. In one embodiment,oligonucleotide sequences, primers, and/or probes that are complimentaryto sequences in the hMPV genome are used to classify the etiologicalagents of hMPV infections into distinct subgroups and/or variants inconjunction with methods known to one skilled in the art, e.g. RT-PCR,PCR, primer run on assays, and various blotting techniques. In onespecific embodiment, a biological sample is used to copy or amplify aspecific segment of the hMPV genome, using RT-PCR. In a furtherembodiment, the sequence of the segment is obtained and compared withknown sequences of hMPV, and the comparison is used to classify the hMPVstrain into a distinct subgroup or variant or to classify the hMPVstrain into a new subgroup or variant. In another embodiment, theinvention relates to diagnostic kits that can be used to distinguishbetween different subgroups and/or variants of hMPV.

In a preferred embodiment, diagnosis and/or treatment of a specificviral infection is performed with reagents that are most specific forthe specific virus causing the infection. In this case this means thatit is preferred that the diagnosis and/or treatment of an MPV infectionis performed with reagents that are most specific for MPV. This by nomeans however excludes the possibility that less specific, butsufficiently cross-reactive reagents are used instead, for examplebecause they are more easily available and sufficiently address the taskat hand. Herein it is for example provided to perform virological and/orserological diagnosis of MPV infections in mammals with reagents derivedfrom APV, in particular with reagents derived from APV-C, in thedetailed description herein it is for example shown that sufficientlytrustworthy serological diagnosis of MPV infections in mammals can beachieved by using an ELISA specifically designed to detect APVantibodies in birds. A particular useful test for this purpose is anELISA test designed for the detection of APV antibodies (e.g. in serumor egg yolk), one commercially available version of which is known asAPV-Ab SVANOVIR® which is manufactured by SVANOVA Biotech AB, UppsalScience Park Glunten SE-751 83 Uppsala Sweden. The reverse situation isalso the case, herein it is for example provided to perform virologicaland/or serological diagnosis of APV infections in mammals with reagentsderived from MPV, in the detailed description herein it is for exampleshown that sufficiently trustworthy serological diagnosis of APVinfections in birds can be achieved by using an ELISA designed to detectMPV antibodies. Considering that antigens and antibodies have alock-and-key relationship, detection of the various antigens can beachieved by selecting the appropriate antibody having sufficientcross-reactivity. Of course, for relying on such cross-reactivity, it isbest to select the reagents (such as antigens or antibodies) underguidance of the amino acid homologies that exist between the various(glyco)proteins of the various viruses, whereby reagents relating to themost homologous proteins will be most useful to be used in tests relyingon the cross-reactivity.

For nucleic acid detection, it is even more straightforward, instead ofdesigning primers or probes based on heterologous nucleic acid sequencesof the various viruses and thus that detect differences between theessentially mammalian or avian Metapneumoviruses, it suffices to designor select primers or probes based on those stretches of virus-specificnucleic acid sequences that show high homology. In general, for nucleicacid sequences, homology percentages of 90% or higher guaranteesufficient cross-reactivity to be relied upon in diagnostic testsutilizing stringent conditions of hybridization.

The invention for example provides a method for virologically diagnosinga MPV infection of an animal, in particular of a mammal, more inparticular of a human being, comprising determining in a sample of theanimal the presence of a viral isolate or component thereof by reactingthe sample with a MPV specific nucleic acid a or antibody according tothe invention, and a method for serologically diagnosing an MPVinfection of a mammal comprising determining in a sample of the mammalthe presence of an antibody specifically directed against an MPV orcomponent thereof by reacting the sample with a MPV-specificproteinaceous molecule or fragment thereof or an antigen according tothe invention. The invention also provides a diagnostic kit fordiagnosing an MPV infection comprising an MPV, an MPV-specific nucleicacid, proteinaceous molecule or fragment thereof, antigen and/or anantibody according to the invention, and preferably a means fordetecting the MPV, MPV-specific nucleic acid, proteinaceous molecule orfragment thereof, antigen and/or an antibody, the means for examplecomprising an excitable group such as a fluorophore or enzymaticdetection system used in the art (examples of suitable diagnostic kitformat comprise IF, ELISA, neutralization assay, RT-PCR assay). Todetermine whether an as yet unidentified virus component or syntheticanalogue thereof such as nucleic acid, proteinaceous molecule orfragment thereof can be identified as MPV-specific, it suffices toanalyze the nucleic acid or amino acid sequence of the component, forexample for a stretch of the nucleic acid or amino acid, preferably ofat least 10, more preferably at least 25, more preferably at least 40nucleotides or amino acids (respectively), by sequence homologycomparison with known MPV sequences and with known non-MPV sequencesAPV-C is preferably used) using for example phylogenetic analyses asprovided herein. Depending on the degree of relationship with the MPV ornon-MPV sequences, the component or synthetic analogue can beidentified.

The invention also provides method for virologically diagnosing an MPVinfection of a mammal comprising determining in a sample of the mammalthe presence of a viral isolate or component thereof by reacting thesample with a cross-reactive nucleic acid derived from APV (preferablyserotype C) or a cross-reactive antibody reactive with the APV, and amethod for serologically diagnosing an MPV infection of a mammalcomprising determining in a sample of the mammal the presence of across-reactive antibody that is also directed against an APV orcomponent thereof by reacting the sample with a proteinaceous moleculeor fragment thereof or an antigen derived from APV. Furthermore, theinvention provides the use of a diagnostic kit initially designed forAVP or AVP-antibody detection for diagnosing an MPV infection, inparticular for detecting the MPV infection in humans.

The invention also provides methods for virologically diagnosing an APVinfection in a bird comprising determining in a sample of the bird thepresence of a viral isolate or component thereof by reacting the samplewith a cross-reactive nucleic acid derived from MPV or a cross-reactiveantibody reactive with the MPV, and a method for serologicallydiagnosing an APV infection of a bird comprising determining in a sampleof the bird the presence of a cross-reactive antibody that is alsodirected against an MPV or component thereof by reacting the sample witha proteinaceous molecule or fragment thereof or an antigen derived fromMPV. Furthermore, the invention provides the use of a diagnostic kitinitially designed for MPV or MPV-antibody detection for diagnosing anAPV infection, in particular for detecting the APV infection in poultrysuch as a chicken, duck or turkey.

For diagnosis as for treatment, use can be made of the high degree ofhomology among different mammalian MPVs and between MPV and otherviruses, such as, e.g., APV, in particular when circumstances at handmake the use of the more homologous approach less straightforward.Vaccinations that cannot wait, such as emergency vaccinations againstMPV infections can for example be performed with vaccine preparationsderived from APV (preferably type C) isolates when a more homologous MPVvaccine is not available, and, vice versa, vaccinations against APVinfections can be contemplated with vaccine preparations derived fromMPV. Also, reverse genetic techniques make it possible to generatechimeric APV-MPV virus constructs that are useful as a vaccine, beingsufficiently dissimilar to field isolates of each of the respectivestrains to be attenuated to a desirable level. Similar reverse genetictechniques will make it also possible to generate chimericparamyxovirus-metapneumovirus constructs, such as RSV-MPV or P13-MPVconstructs for us in a vaccine preparation. Such constructs areparticularly useful as a combination vaccine to combat respiratory tractillnesses.

Since MPV CPE was virtually indistinguishable from that caused by hRSVor hPIV-1 in tMK or other cell cultures, the MPV may have well goneunnoticed until now. tMK (tertiary monkey kidney cells, i.e. MK cells ina third passage in cell culture) are preferably used due to their lowercosts in comparison to primary or secondary cultures. The CPE is, aswell as with some of the classical Paramyxoviridae, characterized bysyncytium formation after which the cells showed rapid internaldisruption, followed by detachment of the cells from the monolayer. Thecells usually (but not always) displayed CPE after three passages ofvirus from original material, at day 10 to 14 post inoculation, somewhatlater than CPE caused by other viruses such as hRSV or hPIV-1.

As an example, the invention provides a not previously identifiedparamyxovirus from nasopharyngeal aspirate samples taken from 28children suffering from severe RTI. The clinical symptoms of thesechildren were largely similar to those caused by hRSV. Twenty-seven ofthe patients were children below the age of five years and half of thesewere between 1 and 12 months old. The other patient was 18 years old.All individuals suffered from upper RTI, with symptoms ranging fromcough, myalgia, vomiting and fever to bronchiolitis and severepneumonia. The majority of these patients were hospitalized for one totwo weeks.

The virus isolates from these patients had the paramyxovirus morphologyin negative contrast electron microscopy but did not react with specificantisera against known human and animal paramyxoviruses. They were allclosely related to one another as determined by indirectimmunofluorescence assays (IFA) with sera raised against two of theisolates. Sequence analyses of nine of these isolates revealed that thevirus is somewhat related to APV. Based on virological data, sequencehomology as well as the genomic organization we propose that the virusis a member of metapneumovirus genus. Serological surveys showed thatthis virus is a relatively common pathogen since the seroprevalence inthe Netherlands approaches 100% of humans by the age of five years.Moreover, the seroprevalence was found to be equally high in seracollected from humans in 1958, indicating this virus has beencirculating in the human population for more than 40 years. Theidentification of this proposed new member of the Metapneumovirus genusnow also provides for the development of means and methods fordiagnostic assays or test kits and vaccines or serum or antibodycompositions for viral respiratory tract infections, and for methods totest or screen for antiviral agents useful in the treatment of MPVinfections.

Methods and means provided herein are particularly useful in adiagnostic kit for diagnosing a MPV infection, be it by virological orserological diagnosis. Such kits or assays may for example comprise avirus, a nucleic acid, a proteinaceous molecule or fragment thereof, anantigen and/or an antibody according to the invention. Use of a virus, anucleic acid, a proteinaceous molecule or fragment thereof, an antigenand/or an antibody according to the invention is also provided for theproduction of a pharmaceutical composition, for example for thetreatment or prevention of MPV infections and/or for the treatment orprevention of respiratory tract illnesses, in particular in humans.Attenuation of the virus can be achieved by established methodsdeveloped for this purpose, including but not limited to the use ofrelated viruses of other species, serial passages through laboratoryanimals or/and tissue/cell cultures, site directed mutagenesis ofmolecular clones and exchange of genes or gene fragments between relatedviruses.

5.15 Compositions of the Invention and Components of MammalianMetapneumovirus

The invention relates to nucleic acid sequences of a mammalian MPV,proteins of a mammalian MPV, and antibodies against proteins of amammalian MPV. The invention further relates to homologs of nucleic acidsequences of a mammalian MPV and homologs of proteins of a mammalianMPV. The invention further relates to nucleic acid sequences encodingfusion proteins, wherein the fusion protein contains a protein of amammalian MPV or a fragment thereof and one or more peptides or proteinsthat are not derived from mammalian MPV. In a specific embodiment, afusion protein of the invention contains a protein of a mammalian MPV ora fragment thereof and a peptide tag, such as, but not limited to apolyhistidine tag. The invention further relates to fusion proteins,wherein the fusion protein contains a protein of a mammalian MPV or afragment thereof and one or more peptides or proteins that are notderived from mammalian MPV. The invention also relates to derivatives ofnucleic acids encoding a protein of a mammalian MPV. The invention alsorelates to derivatives of proteins of a mammalian MPV. A derivative canbe, but is not limited to, mutant forms of the protein, such as, but notlimited to, additions, deletions, truncations, substitutions, andinversions. A derivative can further be a chimeric form of the proteinof the mammalian MPV, wherein at least one domain of the protein isderived from a different protein. A derivative can also be a form of aprotein of a mammalian MPV that is covalently or non-covalently linkedto another molecule, such as, e.g., a drug.

The viral isolate termed NL/1/00 (also 00-1) is a mammalian MPV ofvariant A1 and its genomic sequence is shown in SEQ ID NO:19. The viralisolate termed NL/17/00 is a mammalian MPV of variant A2 and its genomicsequence is shown in SEQ ID NO:20. The viral isolate termed NL/1/99(also 99-1) is a mammalian MPV of variant B1 and its genomic sequence isshown in SEQ ID NO:18. The viral isolate termed NL11/94 is a mammalianMPV of variant B2 and its genomic sequence is shown in SEQ ID NO:21. Alist of sequences disclosed in the present application and thecorresponding SEQ ID NOS is set forth in Table 14.

The protein of a mammalian MPV can be a an N protein, a P protein, a Mprotein, a F protein, a M2-1 protein or a M2-2 protein or a fragmentthereof. A fragment of a protein of a mammalian MPV can be can be atleast 25 amino acids, at least 50 amino acids, at least 75 amino acids,at least 100 amino acids, at least 125 amino acids, at least 150 aminoacids, at least 175 amino acids, at least 200 amino acids, at least 225amino acids, at least 250 amino acids, at least 275 amino acids, atleast 300 amino acids, at least 325 amino acids, at least 350 aminoacids, at least 375 amino acids, at least 400 amino acids, at least 425amino acids, at least 450 amino acids, at least 475 amino acids, atleast 500 amino acids, at least 750 amino acids, at least 1000 aminoacids, at least 1250 amino acids, at least 1500 amino acids, at least1750 amino acids, at least 2000 amino acids or at least 2250 amino acidsin length. A fragment of a protein of a mammalian MPV can be can be atmost 25 amino acids, at most 50 amino acids, at most 75 amino acids, atmost 100 amino acids, at most 125 amino acids, at most 150 amino acids,at most 175 amino acids, at most 200 amino acids, at most 225 aminoacids, at most 250 amino acids, at most 275 amino acids, at most 300amino acids, at most 325 amino acids, at most 350 amino acids, at most375 amino acids, at most 400 amino acids, at most 425 amino acids, atmost 450 amino acids, at most 475 amino acids, at most 500 amino acids,at most 750 amino acids, at most 1000 amino acids, at most 1250 aminoacids, at most 1500 amino acids, at most 1750 amino acids, at most 2000amino acids or at most 2250 amino acids in length.

In certain embodiments of the invention, the protein of a mammalian MPVis a N protein, wherein the N protein is phylogenetically closer relatedto a N protein of a mammalian MPV, such as the N protein encoded by,e.g., the viral genome of SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, orSEQ ID NO:21, (see also Table 14 for a description of the SEQ ID NOS)than it is related to the N protein of APV type C. In certainembodiments of the invention, the protein of a mammalian MPV is a Pprotein, wherein the P protein is phylogenetically closer related to a Pprotein of a mammalian MPV, such as the P protein encoded by, e.g., theviral genome of SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, or SEQ IDNO:21, than it is related to the N protein of APV type C. In certainembodiments of the invention, the protein of a mammalian MPV is a Mprotein, wherein the M protein is closer related to a M protein of amammalian MPV, such as the M protein encoded by, e.g., the viral genomeof SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:21, than it isrelated to the M protein of APV type C. In certain embodiments of theinvention, the protein of a mammalian MPV is a F protein, wherein the Fprotein is phylogenetically closer related to a F protein of a mammalianMPV, such as the F protein encoded by, e.g., the viral genome of SEQ IDNO:18, SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:21, than it is relatedto the F protein of APV type C. In certain embodiments of the invention,the protein of a mammalian MPV is a M2-1 protein, wherein the M2-1protein is phylogenetically closer related to a M2-1 protein of amammalian MPV, such as the M2-1 protein encoded by, e.g., the viralgenome of SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:21,than it is related to the M2-1 protein of APV type C. In certainembodiments of the invention, the protein of a mammalian MPV is a M2-2protein, wherein the M2-2 protein is phylogenetically closer related toa M2-2 protein of a mammalian MPV, such as the M2-2 protein encoded by,e.g., the viral genome of SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, orSEQ ID NO:21, than it is related to the M2-2 protein of APV type C. Incertain embodiments of the invention, the protein of a mammalian MPV isa G protein, wherein the G protein is phylogenetically closer related toa G protein of a mammalian MPV, such as the G protein encoded by, e.g.,the viral genome of SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, or SEQ IDNO:21, than it is related to any protein of APV type C. In certainembodiments of the invention, the protein of a mammalian MPV is a SHprotein, wherein the SH protein is phylogenetically closer related to aSH protein of a mammalian MPV, such as the SH protein encoded by, e.g.,the viral genome of SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, or SEQ IDNO:21, than it is related to any protein of APV type C. In certainembodiments of the invention, the protein of a mammalian MPV is a Lprotein, wherein the L protein is phylogenetically closer related to a Lprotein of a mammalian MPV, such as the SH protein encoded by, e.g., theviral genome of SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, or SEQ IDNO:21, than it is related to any protein of APV type C.

In certain embodiments of the invention, the protein of a mammalian MPVis a N protein, wherein the N protein is at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 98%, at least 99%, or at least 99.5% identical tothe amino acid sequence of a N protein encoded by the viral genome ofSEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:21 (the aminoacid sequences of the respective N proteins are disclosed in SEQ IDNO:366-369; see also Table 14). In certain embodiments of the invention,the protein of a mammalian MPV is a N protein, wherein the P protein isat least 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or atleast 99.5% identical to the amino acid sequence of a P protein encodedby the viral genome of SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, or SEQID NO:21 (the amino acid sequences of the respective P proteins aredisclosed in SEQ ID NO:374-377; see also Table 14). In certainembodiments of the invention, the protein of a mammalian MPV is a Mprotein, wherein the M protein is at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95%, at least 98%, at least 99%, or at least 99.5% identical to theamino acid sequence of a M protein encoded by the viral genome of SEQ IDNO:18, SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:21 (the amino acidsequences of the respective M proteins are disclosed in SEQ IDNO:358-361; see also Table 14). In certain embodiments of the invention,the protein of a mammalian MPV is a F protein, wherein the F protein isat least 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or atleast 99.5% identical to the amino acid sequence of a F protein encodedby the viral genome of SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, or SEQID NO:21 (the amino acid sequences of the respective F proteins aredisclosed in SEQ ID NO:314-317; see also Table 14). In certainembodiments of the invention, the protein of a mammalian MPV is a M2-1protein, wherein the M2-1 protein is at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 98%, at least 99%, or at least 99.5% identical tothe amino acid sequence of a M2-1 protein encoded by the viral genome ofSEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:21 (the aminoacid sequences of the respective M2-1 proteins are disclosed in SEQ IDNO:338-341; see also Table 14). In certain embodiments of the invention,the protein of a mammalian MPV is a M2-2 protein, wherein the M2-2protein is at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 98%, atleast 99%, or at least 99.5% identical to the amino acid sequence of aM2-2 protein encoded by the viral genome of SEQ ID NO:18, SEQ ID NO:19,SEQ ID NO:20, or SEQ ID NO:21 (the amino acid sequences of therespective M2-2 proteins are disclosed in SEQ ID NO:346-349; see alsoTable 14). In certain embodiments of the invention, the protein of amammalian MPV is a G protein, wherein the G protein is at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%identical to the amino acid sequence of a G protein encoded by the viralgenome of SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:21 (theamino acid sequences of the respective G proteins are disclosed in SEQID NO:322-325; see also Table 14). In certain embodiments of theinvention, the protein of a mammalian MPV is a SH protein, wherein theSH protein is at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 98%, atleast 99%, or at least 99.5% identical to the amino acid sequence of aSH protein encoded by the viral genome of SEQ ID NO:18, SEQ ID NO:19,SEQ ID NO:20, or SEQ ID NO:21 (the amino acid sequences of therespective SH proteins are disclosed in SEQ ID NO:382-385; see alsoTable 14). In certain embodiments of the invention, the protein of amammalian MPV is a L protein, wherein the L protein is at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%identical to the amino acid sequence of a L protein encoded by the viralgenome of SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:21 (theamino acid sequences of the respective L proteins are disclosed in SEQID NO:330-333; see also Table 14).

A fragment of a protein of mammalian MPV is at least 60%, at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 98%, at least 99%, or at least 99.5% identical tothe homologous protein encoded by the virus of SEQ ID NO:18, SEQ IDNO:19, SEQ ID NO:20, or SEQ ID NO:21 over the portion of the proteinthat is homologous to the fragment. In a specific, illustrativeembodiment, the invention provides a fragment of the F protein of amammalian MPV that contains the ectodomain of the F protein and homologsthereof. The homolog of the fragment of the F protein that contains theectodomain is at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 98%, atleast 99%, or at least 99.5% identical to the corresponding fragmentcontaining the ectodomain of the F protein encoded by a virus of SEQ IDNO:18, SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:21 (the amino acidsequences of the respective F proteins are disclosed in SEQ IDNO:314-317; see also Table 14).

In certain embodiments, the invention provides a protein of a mammalianMPV of subgroup A and fragments thereof. The invention provides a Nprotein of a mammalian MPV of subgroup A, wherein the N protein isphylogenetically closer related to the N protein encoded by a virus ofSEQ ID NO:19 or SEQ ID NO:20 than it is related to the N protein encodedby a virus encoded by SEQ ID NO:18 or SEQ ID NO:21. The inventionprovides a G protein of a mammalian MPV of subgroup A, wherein the Gprotein is phylogenetically closer related to the G protein encoded by avirus of SEQ ID NO:19 or SEQ ID NO:20 than it is related to the Gprotein encoded by a virus encoded by SEQ NO:18 or SEQ ID NO:21. Theinvention provides a P protein of a mammalian MPV of subgroup A, whereinthe P protein is phylogenetically closer related to the P proteinencoded by a virus of SEQ ID NO:19 or SEQ ID NO:20 than it is related tothe P protein encoded by a virus encoded by SEQ ID NO:18 or SEQ IDNO:21. The invention provides a M protein of a mammalian MPV of subgroupA, wherein the M protein is phylogenetically closer related to the Mprotein encoded by a virus of SEQ ID NO:19 or SEQ ID NO:20 than it isrelated to the M protein encoded by a virus encoded by SEQ ID NO:18 orSEQ ID NO:21. The invention provides a N protein of a mammalian MPV ofsubgroup A, wherein the F protein is phylogenetically closer related tothe F protein encoded by a virus of SEQ ID NO:19 or SEQ ID NO:20 than itis related to the F protein encoded by a virus encoded by SEQ ID NO:18or SEQ ID NO:21. The invention provides a M2-1 protein of a mammalianMPV of subgroup A, wherein the M2-1 protein is phylogenetically closerrelated to the M2-1 protein encoded by a virus of SEQ ID NO:19 or SEQ IDNO:20 than it is related to the M2-1 protein encoded by a virus encodedby SEQ ID NO:18 or SEQ ID NO:21. The invention provides a M2-2 proteinof a mammalian MPV of subgroup A, wherein the M2-2 protein isphylogenetically closer related to the M2-2 protein encoded by a virusof SEQ ID NO:19 or SEQ ID NO:20 than it is related to the M2-2 proteinencoded by a virus encoded by SEQ ID NO:18 or SEQ ID NO:21. Theinvention provides a SH protein of a mammalian MPV of subgroup A,wherein the SH protein is phylogenetically closer related to the SHprotein encoded by a virus of SEQ ID NO:19 or SEQ ID NO:20 than it isrelated to the SH protein encoded by a virus encoded by SEQ ID NO:18 orSEQ ID NO:21. The invention provides a L protein of a mammalian MPV ofsubgroup A, wherein the L protein is phylogenetically closer related tothe L protein encoded by a virus of SEQ ID NO:19 or SEQ ID NO:20 than itis related to the L protein encoded by a virus encoded by SEQ ID NO:18or SEQ ID NO:21.

In other embodiments, the invention provides a protein of a mammalianMPV of subgroup B or fragments thereof. The invention provides a Nprotein of a mammalian MPV of subgroup B, wherein the N protein isphylogenetically closer related to the N protein encoded by a virus ofSEQ ID NO:18 or SEQ ID NO:21 than it is related to the N protein encodedby a virus encoded by SEQ ID NO:19 or SEQ ID NO:20. The inventionprovides a G protein of a mammalian MPV of subgroup A, wherein the Gprotein is phylogenetically closer related to the G protein encoded by avirus of SEQ ID NO:18 or SEQ ID NO:21 than it is related to the Gprotein encoded by a virus encoded by SEQ ID NO:19 or SEQ ID NO:20. Theinvention provides a P protein of a mammalian MPV of subgroup A, whereinthe P protein is phylogenetically closer related to the P proteinencoded by a virus of SEQ ID NO:18 or SEQ ID NO:21 than it is related tothe P protein encoded by a virus encoded by SEQ ID NO:19 or SEQ IDNO:20. The invention provides a M protein of a mammalian MPV of subgroupA, wherein the M protein is phylogenetically closer related to the Mprotein encoded by a virus of SEQ ID NO:18 or SEQ ID NO:21 than it isrelated to the M protein encoded by a virus encoded by SEQ ID NO:19 orSEQ ID NO:20. The invention provides a N protein of a mammalian MPV ofsubgroup A, wherein the F protein is phylogenetically closer related tothe F protein encoded by a virus of SEQ ID NO:18 or SEQ ID NO:21 than itis related to the F protein encoded by a virus encoded by SEQ ID NO:19or SEQ ID NO:20. The invention provides a M2-1 protein of a mammalianMPV of subgroup A, wherein the M2-1 protein is phylogenetically closerrelated to the M2-1 protein encoded by a virus of SEQ ID NO:18 or SEQ IDNO:21 than it is related to the M2-1 protein encoded by a virus encodedby SEQ ID NO:19 or SEQ ID NO:20. The invention provides a M2-2 proteinof a mammalian MPV of subgroup A, wherein the M2-2 protein isphylogenetically closer related to the M2-2 protein encoded by a virusof SEQ ID NO:18 or SEQ ID NO:21 than it is related to the M2-2 proteinencoded by a virus encoded by SEQ ID NO:19 or SEQ ID NO:20. Theinvention provides a SH protein of a mammalian MPV of subgroup A,wherein the SH protein is phylogenetically closer related to the SHprotein encoded by a virus of SEQ ID NO:18 or SEQ ID NO:21 than it isrelated to the SH protein encoded by a virus encoded by SEQ ID NO:19 orSEQ ID NO:20. The invention provides a L protein of a mammalian MPV ofsubgroup A, wherein the L protein is phylogenetically closer related tothe L protein encoded by a virus of SEQ ID NO:18 or SEQ ID NO:21 than itis related to the L protein encoded by a virus encoded by SEQ ID NO:19or SEQ ID NO:20.

The invention further provides proteins of a mammalian MPV of variantA1, A2, B1 or B2. In certain embodiments of the invention, the proteinsof the different variants of mammalian MPV can be distinguished fromeach other by way of their amino acid sequence identities (see, e.g.,FIG. 42B). A variant of mammalian MPV can be, but is not limited to, A1,A2, B1 or B2. The invention, however, also contemplates isolates ofmammalian MPV that are members of another variant.

The invention provides a G protein of a mammalian MPV variant B1,wherein the G protein of a mammalian MPV variant B1 is phylogeneticallycloser related to the G protein of the prototype of variant B1, isolateNL/1/99, than it is related to the G protein of the prototype of variantA1, isolate NL/1/00, the G protein of the prototype of A2, isolateNL/17/00, or the G protein of the prototype of B2, isolate NL/1/94. Theinvention provides a G protein of a mammalian MPV variant B1, whereinthe amino acid sequence of the G protein is at least 66%, at least 70%,at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, or at least 99% or at least 99.5% identical to the G proteinof a mammalian MPV variant B1 as represented by the prototype NL/1/99(SEQ ID NO:324). The invention provides a N protein of a mammalian MPVvariant B1, wherein the N protein of a mammalian MPV variant B1 isphylogenetically closer related to the N protein of the prototype ofvariant B1, isolate NL/1/99, than it is related to the N protein of theprototype of variant A1, isolate NL/1/00, the N protein of the prototypeof A2, isolate NL/17/00, or the N protein of the prototype of B2,isolate NL/1/94. The invention provides a N protein of a mammalian MPVvariant B1, wherein the amino acid sequence of the N protein is at least98.5% or at least 99% or at least 99.5% identical to the N protein of amammalian MPV variant B1 as represented by the prototype NL/1/99 (SEQ IDNO:368). The invention provides a P protein of a mammalian MPV variantB1, wherein the P protein of a mammalian MPV variant B1 isphylogenetically closer related to the P protein of the prototype ofvariant B1, isolate NL/1/99, than it is related to the P protein of theprototype of variant A1, isolate NL/1/00, the P protein of the prototypeof A2, isolate NL/17/00, or the P protein of the prototype of B2,isolate NL/1/94. The invention provides a P protein of a mammalian MPVvariant B1, wherein the amino acid sequence of the P protein is at least96%, at least 98%, or at least 99% or at least 99.5% identical the Pprotein of a mammalian MPV variant B1 as represented by the prototypeNL/1/99 (SEQ ID NO:376). The invention provides a M protein of amammalian MPV variant B1, wherein the M protein of a mammalian MPVvariant B1 is phylogenetically closer related to the M protein of theprototype of variant B1, isolate NL/1/99, than it is related to the Mprotein of the prototype of variant A1, isolate NL/1/00, the M proteinof the prototype of A2, isolate NL/17/00, or the M protein of theprototype of B2, isolate NL/1/94. The invention provides a M protein ofa mammalian MPV variant B1, wherein the amino acid sequence of the Mprotein is identical the M protein of a mammalian MPV variant B1 asrepresented by the prototype NL/1/99 (SEQ ID NO:360). The inventionprovides a F protein of a mammalian MPV variant B1, wherein the Fprotein of a mammalian MPV variant B1 is phylogenetically closer relatedto the F protein of the prototype of variant B1, isolate NL/1/99, thanit is related to the F protein of the prototype of variant A1, isolateNL/1/00, the F protein of the prototype of A2, isolate NL/17/00, or theF protein of the prototype of B2, isolate NL/1/94. The inventionprovides a F protein of a mammalian MPV variant B1, wherein the aminoacid sequence of the F protein is at least 99% identical to the Fprotein of a mammalian MPV variant B1 as represented by the prototypeNL/1/99 (SEQ ID NO:316). The invention provides a M2-1 protein of amammalian MPV variant B1, wherein the M2-1 protein of a mammalian MPVvariant B1 is phylogenetically closer related to the M2-1 protein of theprototype of variant B1, isolate NL/1/99, than it is related to the M2-1protein of the prototype of variant A1, isolate NL/1/00, the M2-1protein of the prototype of A2, isolate NL/17/00, or the M2-1 protein ofthe prototype of B2, isolate NL/1/94. The invention provides a M2-1protein of a mammalian MPV variant B1, wherein the amino acid sequenceof the M2-1 protein is at least 98% or at least 99% or at least 99.5%identical the M2-1 protein of a mammalian MPV variant B1 as representedby the prototype NL/1/99 (SEQ ID NO:340). The invention provides a M2-2protein of a mammalian MPV variant B1, wherein the M2-2 protein of amammalian MPV variant B1 is phylogenetically closer related to the M2-2protein of the prototype of variant B1, isolate NL/1/99, than it isrelated to the M2-2 protein of the prototype of variant A1, isolateNL/00, the M2-2 protein of the prototype of A2, isolate NL/17/00, or theM2-2 protein of the prototype of B2, isolate NL/1/94. The inventionprovides a M2-2 protein of a mammalian MPV variant B1, wherein the aminoacid sequence of the M2-2 protein is at least 99% or at least 99.5%identical the M2-2 protein of a mammalian MPV variant B1 as representedby the prototype NL/1/99 (SEQ ID NO:348). The invention provides a SHprotein of a mammalian MPV variant B1, wherein the SH protein of amammalian MPV variant B1 is phylogenetically closer related to the SHprotein of the prototype of variant B1, isolate NL/1/99, than it isrelated to the SH protein of the prototype of variant A1, isolateNL/1/00, the SH protein of the prototype of A2, isolate NL/17/00, or theSH protein of the prototype of B2, isolate NL/1/94. The inventionprovides a SH protein of a mammalian MPV variant B1, wherein the aminoacid sequence of the SH protein is at least 83%, at least 85%, at least90%, at least 95%, at least 98%, or at least 99% or at least 99.5%identical the SH protein of a mammalian MPV variant B1 as represented bythe prototype NL/1/99 (SEQ ID NO:384). The invention provides a Lprotein of a mammalian MPV variant B1, wherein the L protein of amammalian MPV variant B1 is phylogenetically closer related to the Lprotein of the prototype of variant B1, isolate NL/1/99, than it isrelated to the L protein of the prototype of variant A1, isolateNL/1/00, the L protein of the prototype of A2, isolate NL/17/00, or theL protein of the prototype of B2, isolate NL/1/94. The inventionprovides a L protein of a mammalian MPV variant B1, wherein the aminoacid sequence of the L protein is at least 99% or at least 99.5%identical the L protein a mammalian MPV variant B1 as represented by theprototype NL/1/99 (SEQ ID NO:332).

The invention provides a G protein of a mammalian MPV variant A1,wherein the G protein of a mammalian MPV variant A1 is phylogeneticallycloser related to the G protein of the prototype of variant A1, isolateNL/1/00, than it is related to the G protein of the prototype of variantB1, isolate NL/1/99, the G protein of the prototype of A2, isolateNL/17/00, or the G protein of the prototype of B2, isolate NL/1/94. Theinvention provides a G protein of a mammalian MPV variant A1, whereinthe amino acid sequence of the G protein is at least 66%, at least 70%,at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, or at least 99% or at least 99.5% identical to the G proteinof a mammalian MPV variant A1 as represented by the prototype NL/1/00(SEQ ID NO:322). The invention provides a N protein of a mammalian MPVvariant A1, wherein the N protein of a mammalian MPV variant A1 isphylogenetically closer related to the N protein of the prototype ofvariant A1, isolate NL/1/00, than it is related to the N protein of theprototype of variant B1, isolate NL/1/99, the N protein of the prototypeof A2, isolate NL/17/00, or the N protein of the prototype of B2,isolate NL/1/94. The invention provides a N protein of a mammalian MPVvariant A1, wherein the amino acid sequence of the N protein is at least99.5% identical to the N protein of a mammalian MPV variant A1 asrepresented by the prototype NL/1/00 (SEQ ID NO:366). The inventionprovides a P protein of a mammalian MPV variant A1, wherein the Pprotein of a mammalian MPV variant A1 is phylogenetically closer relatedto the P protein of the prototype of variant A1, isolate NL/1/00, thanit is related to the P protein of the prototype of variant B1, isolateNL/1/99, the P protein of the prototype of A2, isolate NL/17/00, or theP protein of the prototype of B2, isolate NL/1/94. The inventionprovides a P protein of a mammalian MPV variant A1, wherein the aminoacid sequence of the P protein is at least 96%, at least 98%, or atleast 99% or at least 99.5% identical to the P protein of a mammalianMPV variant A1 as represented by the prototype NL/1/00 (SEQ ID NO:374).The invention provides a M protein of a mammalian MPV variant A1,wherein the M protein of a mammalian MPV variant A1 is phylogeneticallycloser related to the M protein of the prototype of variant A1, isolateNL/1/00, than it is related to the M protein of the prototype of variantB1, isolate NL/1/99, the M protein of the prototype of A2, isolateNL/17/00, or the M protein of the prototype of B2, isolate NL/1/94. Theinvention provides a M protein of a mammalian MPV variant A1, whereinthe amino acid sequence of the M protein is at least 99% or at least99.5% identical to the M protein of a mammalian MPV variant A1 asrepresented by the prototype NL/1/00 (SEQ ID NO:358). The inventionprovides a F protein of a mammalian MPV variant A1, wherein the Fprotein of a mammalian MPV variant A1 is phylogenetically closer relatedto the F protein of the prototype of variant A1, isolate NL/1/00, thanit is related to the F protein of the prototype of variant B1, isolateNL/1/99, the F protein of the prototype of A2, isolate NL/17/00, or theF protein of the prototype of B2, isolate NL/1/94. The inventionprovides a F protein of a mammalian MPV variant A1, wherein the aminoacid sequence of the F protein is at least 98% or at least 99% or atleast 99.5% identical to the F protein of a mammalian MPV variant A1 asrepresented by the prototype NL/1/00 (SEQ ID NO:314). The inventionprovides a M2-1 protein of a mammalian MPV variant A1, wherein the M2-1protein of a mammalian MPV variant A1 is phylogenetically closer relatedto the M2-1 protein of the prototype of variant A1, isolate NL/1/00,than it is related to the M2-1 protein of the prototype of variant B1,isolate NL/1/99, the M2-1 protein of the prototype of A2, isolateNL/17/00, or the M2-1 protein of the prototype of B2, isolate NL/1/94.The invention provides a M2-1 protein of a mammalian MPV variant A1,wherein the amino acid sequence of the M2-1 protein is at least 99% orat least 99.5% identical to the M2-1 protein of a mammalian MPV variantA1 as represented by the prototype NL/1/00 (SEQ ID NO:338). Theinvention provides a M2-2 protein of a mammalian MPV variant A1, whereinthe M2-2 protein of a mammalian MPV variant A1 is phylogeneticallycloser related to the M2-2 protein of the prototype of variant A1,isolate NL/1/00, than it is related to the M2-2 protein of the prototypeof variant B1, isolate NL/1/99, the M2-2 protein of the prototype of A2,isolate NL/17/00, or the M2-2 protein of the prototype of B2, isolateNL/1/94. The invention provides a M2-2 protein of a mammalian MPVvariant A1, wherein the amino acid sequence of the M2-2 protein is atleast 96% or at least 99% or at least 99.5% identical to the M2-2protein of a mammalian MPV variant A1 as represented by the prototypeNL/1/00 (SEQ ID NO:346). The invention provides a SH protein of amammalian MPV variant A1, wherein the SH protein of a mammalian MPVvariant A1 is phylogenetically closer related to the SH protein of theprototype of variant A1, isolate NL/1/00, than it is related to the SHprotein of the prototype of variant B1, isolate NL/1/99, the SH proteinof the prototype of A2, isolate NL/17/00, or the SH protein of theprototype of B2, isolate NL/1/94. The invention provides a SH protein ofa mammalian MPV variant A1, wherein the amino acid sequence of the SHprotein is at least 84%, at least 90%, at least 95%, at least 98%, or atleast 99% or at least 99.5% identical to the SH protein of a mammalianMPV variant A1 as represented by the prototype NL/1/00 (SEQ ID NO:382).The invention provides a L protein of a mammalian MPV variant A1,wherein the L protein of a mammalian MPV variant A1 is phylogeneticallycloser related to the L protein of the prototype of variant A1, isolateNL/1/00, than it is related to the L protein of the prototype of variantB1, isolate NL/1/99, the L protein of the prototype of A2, isolateNL/17/00, or the L protein of the prototype of B2, isolate NL/1/94. Theinvention provides a L protein of a mammalian MPV variant A1, whereinthe amino acid sequence of the L protein is at least 99% or at least99.5% identical to the L protein of a virus of a mammalian MPV variantA1 as represented by the prototype NL/1/00 (SEQ ID NO:330).

The invention provides a G protein of a mammalian MPV variant A2,wherein the G protein of a mammalian MPV variant A2 is phylogeneticallycloser related to the G protein of the prototype of variant A2, isolateNL/17/00, than it is related to the G protein of the prototype ofvariant B1, isolate NL/1/99, the G protein of the prototype of A1,isolate NL/1/00, or the G protein of the prototype of B2, isolateNL/1/94. The invention provides a G protein of a mammalian MPV variantA2, wherein the amino acid sequence of the G protein is at least 66%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 98%, at least 99% or at least 99.5% identical to theG protein of a mammalian MPV variant A2 as represented by the prototypeNL/17/00 (SEQ ID NO:332). The invention provides a N protein of amammalian MPV variant A2, wherein the N protein of a mammalian MPVvariant A2 is phylogenetically closer related to the N protein of theprototype of variant A2, isolate NL/17/00, than it is related to the Nprotein of the prototype of variant B1, isolate NL/1/99, the N proteinof the prototype of A1, isolate NL/1/00, or the N protein of theprototype of B2, isolate NL/1/94. The invention provides a N protein ofa mammalian MPV variant A2, wherein the amino acid sequence of the Nprotein at least 99.5% identical to the N protein of a mammalian MPVvariant A2 as represented by the prototype NL/17/00 (SEQ ID NO:367). Theinvention provides a P protein of a mammalian MPV variant A2, whereinthe P protein of a mammalian MPV variant A2 is phylogenetically closerrelated to the P protein of the prototype of variant A2, isolateNL/17/00, than it is related to the P protein of the prototype ofvariant B1, isolate NL/1/99, the P protein of the prototype of A1,isolate NL/1/00, or the P protein of the prototype of B2, isolateNL/1/94. The invention provides a P protein of a mammalian MPV variantA2, wherein the amino acid sequence of the P protein is at least 96%, atleast 98%, at least 99% or at least 99.5% identical to the P protein ofa mammalian MPV variant A2 as represented by the prototype NL/17/00 (SEQID NO:375). The invention provides a M protein of a mammalian MPVvariant A2, wherein the M protein of a mammalian MPV variant A2 isphylogenetically closer related to the M protein of the prototype ofvariant A2, isolate NL/17/00, than it is related to the M protein of theprototype of variant B1, isolate NL/1/99, the M protein of the prototypeof A1, isolate NL/1/00, or the M protein of the prototype of B2, isolateNL/1/94. The invention provides a M protein of a mammalian MPV variantA2, wherein the amino acid sequence of the M protein is at least 99%, orat least 99.5% identical to the M protein of a mammalian MPV variant A2as represented by the prototype NL/17/00 (SEQ ID NO:359). The inventionprovides a F protein of a mammalian MPV variant A2, wherein the Fprotein of a mammalian MPV variant A2 is phylogenetically closer relatedto the F protein of the prototype of variant A2, isolate NL/17/00, thanit is related to the F protein of the prototype of variant B1, isolateNL/1/99, the F protein of the prototype of A1, isolate NL/1/00, or the Fprotein of the prototype of B2, isolate NL/1/94. The invention providesa F protein of a mammalian MPV variant A2, wherein the amino acidsequence of the F protein is at least 98%, at least 99% or at least99.5% identical to the F protein of a mammalian MPV variant A2 asrepresented by the prototype NL/17/00 (SEQ ID NO:315). The inventionprovides a M2-1 protein of a mammalian MPV variant A2, wherein the M2-1protein of a mammalian MPV variant A2 is phylogenetically closer relatedto the M2-1 protein of the prototype of variant A2, isolate NL/17/00,than it is related to the M2-1 protein of the prototype of variant B1,isolate NL/1/99, the M2-1 protein of the prototype of A1, isolateNL/1/00, or the M2-1 protein of the prototype of B2, isolate NL/1/94.The invention provides a M2-1 protein of a mammalian MPV variant A2,wherein the amino acid sequence of the M2-1 protein is at least 99%, orat least 99.5% identical to the M2-1 protein of a mammalian MPV variantA2 as represented by the prototype NL/17/00 (SEQ ID NO:339). Theinvention provides a M2-2 protein of a mammalian MPV variant A2, whereinthe M2-2 protein of a mammalian MPV variant A2 is phylogeneticallycloser related to the M2-2 protein of the prototype of variant A2,isolate NL/17/00, than it is related to the M2-2 protein of theprototype of variant B1, isolate NL/1/99, the M2-2 protein of theprototype of A1, isolate NL/1/00, or the M2-2 protein of the prototypeof B2, isolate NL/1/94. The invention provides a M2-2 protein of amammalian MPV variant A2, wherein the amino acid sequence of the M2-2protein is at least 96%, at least 98%, at least 99% or at least 99.5%identical to the M2-2 protein of a mammalian MPV variant A2 asrepresented by the prototype NL/17/00 (SEQ ID NO:347). The inventionprovides a SH protein of a mammalian MPV variant A2, wherein the SHprotein of a mammalian MPV variant A2 is phylogenetically closer relatedto the SH protein of the prototype of variant A2, isolate NL/17/00, thanit is related to the SH protein of the prototype of variant B1, isolateNL/1/99, the SH protein of the prototype of A1, isolate NL/1/00, or theSH protein of the prototype of B2, isolate NL/1/94. The inventionprovides a SH protein of a mammalian MPV variant A2, wherein the aminoacid sequence of the SH protein is at least 84%, at least 85%, at least90%, at least 95%, at least 98%, at least 99% or at least 99.5%identical to the SH protein of a mammalian MPV variant A2 as representedby the prototype NL/17/00 (SEQ ID NO:383). The invention provides a Lprotein of a mammalian MPV variant A2, wherein the L protein of amammalian MPV variant A2 is phylogenetically closer related to the Lprotein of the prototype of variant A2, isolate NL/17/00, than it isrelated to the L protein of the prototype of variant B1, isolateNL/1/99, the L protein of the prototype of A1, isolate NL/1/00, or the Lprotein of the prototype of B2, isolate NL/1/94. The invention providesa L protein of a mammalian MPV variant A2, wherein the amino acidsequence of the L protein is at least 99% or at least 99.5% identical tothe L protein of a mammalian MPV variant A2 as represented by theprototype NL/17/00 (SEQ ID NO:331).

The invention provides a G protein of a mammalian MPV variant B2,wherein the G protein of a mammalian MPV variant B2 is phylogeneticallycloser related to the G protein of the prototype of variant B2, isolateNL/1/94, than it is related to the G protein of the prototype of variantB1, isolate NL/1/99, the G protein of the prototype of A1, isolateNL/1/00, or the G protein of the prototype of A2, isolate NL/17/00. Theinvention provides a G protein of a mammalian MPV variant B2, whereinthe amino acid sequence of the G protein is at least 66%, at least 70%,at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, or at least 99% or at least 99.5% identical to the G proteinof a mammalian MPV variant B2 as represented by the prototype NL/1/94(SEQ ID NO:325). The invention provides a N protein of a mammalian MPVvariant B2, wherein the N protein of a mammalian MPV variant B2 isphylogenetically closer related to the N protein of the prototype ofvariant B2, isolate NL/1/94, than it is related to the N protein of theprototype of variant B1, isolate NL/1/99, the N protein of the prototypeof A1, isolate NL/1/00, or the N protein of the prototype of A2, isolateNL/17/00. The invention provides a N protein of a mammalian MPV variantB2, wherein the amino acid sequence of the N protein is at least 99% orat least 99.5% identical to the N protein of a mammalian MPV variant B2as represented by the prototype NL/1/94 (SEQ ID NO:369). The inventionprovides a P protein of a mammalian MPV variant B2, wherein the Pprotein of a mammalian MPV variant B2 is phylogenetically closer relatedto the P protein of the prototype of variant B2, isolate NL/1/94, thanit is related to the P protein of the prototype of variant B1, isolateNL/1/99, the P protein of the prototype of A1, isolate NL/1/00, or the Pprotein of the prototype of A2, isolate NL/17/00. The invention providesa P protein of a mammalian MPV variant B2, wherein the amino acidsequence of the P protein is at least 96%, at least 98%, or at least 99%or at least 99.5% identical to the P protein of a mammalian MPV variantB2 as represented by the prototype NL/1/94 (SEQ ID NO:377). Theinvention provides a M protein of a mammalian MPV variant B2, whereinthe M protein of a mammalian MPV variant B2 is phylogenetically closerrelated to the M protein of the prototype of variant B2, isolateNL/1/94, than it is related to the M protein of the prototype of variantB1, isolate NL/1/99, the M protein of the prototype of A1, isolateNL/1/00, or the M protein of the prototype of A2, isolate NL/17/00. Theinvention provides a M protein of a mammalian MPV variant B2, whereinthe amino acid sequence of its M protein is identical to the M proteinof a mammalian MPV variant B2 as represented by the prototype NL/1/94(SEQ ID NO:361). The invention provides a F protein of a mammalian MPVvariant B2, wherein the F protein of a mammalian MPV variant B2 isphylogenetically closer related to the F protein of the prototype ofvariant B2, isolate NL/1/94, than it is related to the F protein of theprototype of variant B1, isolate NL/1/99, the F protein of the prototypeof A1, isolate NL/1/00, or the F protein of the prototype of A2, isolateNL/17/00. The invention provides a F protein of a mammalian MPV variantB2, wherein the amino acid sequence of the F protein is at least 99% orat least 99.5% identical to the F protein of a mammalian MPV variant B2as represented by the prototype NL/1/94 (SEQ ID NO:317). The inventionprovides a M2-1 protein of a mammalian MPV variant B2, wherein the M2-1protein of a mammalian MPV variant B2 is phylogenetically closer relatedto the M2-1 protein of the prototype of variant B2, isolate NL/1/94,than it is related to the M2-1 protein of the prototype of variant B1,isolate NL/1/99, the M2-1 protein of the prototype of A1, isolateNL/1/00, or the M2-1 protein of the prototype of A2, isolate NL/17/00.The invention provides a M2-1 protein of a mammalian MPV variant B2,wherein the amino acid sequence of the M2-1 protein is at least 98% orat least 99% or at least 99.5% identical to the M2-1 protein of amammalian MPV variant B2 as represented by the prototype NL/1/94 (SEQ IDNO:341). The invention provides a M2-2 protein of a mammalian MPVvariant B2, wherein the M2-2 protein of a mammalian MPV variant B2 isphylogenetically closer related to the M2-2 protein of the prototype ofvariant B2, isolate NL/1/94, than it is related to the M2-2 protein ofthe prototype of variant B1, isolate NL/1/99, the M2-2 protein of theprototype of A1, isolate NL/1/00, or the M2-2 protein of the prototypeof A2, isolate NL/17/00. The invention provides a M2-2 protein of amammalian MPV variant B2, wherein the amino acid sequence is at least99% or at least 99.5% identical to the M2-2 protein of a mammalian MPVvariant B2 as represented by the prototype NL/1/94 (SEQ ID NO:350). Theinvention provides a SH protein of a mammalian MPV variant B2, whereinthe SH protein of a mammalian MPV variant B2 is phylogenetically closerrelated to the SH protein of the prototype of variant B2, isolateNL/1/94, than it is related to the SH protein of the prototype ofvariant B1, isolate NL/1/99, the SH protein of the prototype of A1,isolate NL/1/00, or the SH protein of the prototype of A2, isolateNL/17/00. The invention provides a SH protein of a mammalian MPV variantB2, wherein the amino acid sequence of the SH protein is at least 84%,at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%or at least 99.5% identical to the SH protein of a mammalian MPV variantB2 as represented by the prototype NL/1/94 (SEQ ID NO:385). Theinvention provides a L protein of a mammalian MPV variant B2, whereinthe L protein of a mammalian MPV variant B2 is phylogenetically closerrelated to the L protein of the prototype of variant B2, isolateNL/1/94, than it is related to the L protein of the prototype of variantB1, isolate NL/1/99, the L protein of the prototype of A1, isolateNL/1/00, or the L protein of the prototype of A2, isolate NL/17/00. Theinvention provides a L protein of a mammalian MPV variant B2, whereinthe and/or if the amino acid sequence of the L protein is at least 99%or at least 99.5% identical to the L protein of a mammalian MPV variantB2 as represented by the prototype NL/1/94 (SEQ ID NO:333).

In certain embodiments, the percentage of sequence identity is based onan alignment of the full length proteins. In other embodiments, thepercentage of sequence identity is based on an alignment of contiguousamino acid sequences of the proteins, wherein the amino acid sequencescan be 25 amino acids, 50 amino acids, 75 amino acids, 100 amino acids,125 amino acids, 150 amino acids, 175 amino acids, 200 amino acids, 225amino acids, 250 amino acids, 275 amino acids, 300 amino acids, 325amino acids, 350 amino acids, 375 amino acids, 400 amino acids, 425amino acids, 450 amino acids, 475 amino acids, 500 amino acids, 750amino acids, 1000 amino acids, 1250 amino acids, 1500 amino acids, 1750amino acids, 2000 amino acids or 2250 amino acids in length.

In certain, specific embodiments, the invention provides a G protein ofa mammalian MPV wherein the G protein has one of the amino acidsequences set forth in SEQ ID NOS:119-153; SEQ ID NOS:322-325 or afragment thereof. In certain, specific embodiments, the inventionprovides a F protein of a mammalian MPV wherein the F protein has one ofthe amino acid sequences set forth in SEQ ID NOS:234-317. In certain,specific embodiments, the invention provides a L protein of a mammalianMPV wherein the L protein has one of the amino acid sequences set forthin SEQ ID NOS:330-333 or a fragment thereof. In certain, specificembodiments, the invention provides a M2-1 protein of a mammalian MPVwherein the M2-1 protein has one of the amino acid sequences set forthin SEQ ID NOS:338-341 or a fragment thereof. In certain, specificembodiments, the invention provides a M2-2 protein of a mammalian MPVwherein the M2-2 protein has one of the amino acid sequences set forthin SEQ ID NOS:346-349 or a fragment thereof. In certain, specificembodiments, the invention provides a M protein of a mammalian MPVwherein the M protein has one of the amino acid sequences set forth inSEQ ID NOS:358-361 or a fragment thereof. In certain, specificembodiments, the invention provides a N protein of a mammalian MPVwherein the N protein has one of the amino acid sequences set forth inSEQ ID NOS:366-369 or a fragment thereof. In certain, specificembodiments, the invention provides a P protein of a mammalian MPVwherein the P protein has one of the amino acid sequences set forth inSEQ ID NOS:374-377 or a fragment thereof. In certain, specificembodiments, the invention provides a SH protein of a mammalian MPVwherein the SH protein has one of the amino acid sequences set forth inSEQ ID NOS:382-385 or a fragment thereof.

In certain embodiments of the invention, a fragment is at least 25 aminoacids, 50 amino acids, 75 amino acids, 100 amino acids, 125 amino acids,150 amino acids, 175 amino acids, 200 amino acids, 225 amino acids, 250amino acids, 275 amino acids, 300 amino acids, 325 amino acids, 350amino acids, 375 amino acids, 400 amino acids, 425 amino acids, 450amino acids, 475 amino acids, 500 amino acids, 750 amino acids, 1000amino acids, 1250 amino acids, 1500 amino acids, 1750 amino acids, 2000amino acids or 2250 amino acids in length. In certain embodiments of theinvention, a fragment is at most 25 amino acids, 50 amino acids, 75amino acids, 100 amino acids, 125 amino acids, 150 amino acids, 175amino acids, 200 amino acids, 225 amino acids, 250 amino acids, 275amino acids, 300 amino acids, 325 amino acids, 350 amino acids, 375amino acids, 400 amino acids, 425 amino acids, 450 amino acids, 475amino acids, 500 amino acids, 750 amino acids, 1000 amino acids, 1250amino acids, 1500 amino acids, 1750 amino acids, 2000 amino acids or2250 amino acids in length.

The invention further provides nucleic acid sequences derived from amammalian MPV. The invention also provides derivatives of nucleic acidsequences derived from a mammalian MPV. In certain specific embodimentsthe nucleic acids are modified.

In certain embodiments, a nucleic acid of the invention encodes a Gprotein, a N protein, a P protein, a M protein, a F protein, a M2-1protein, a M2-2 protein, a SH protein, or a L protein of a mammalian MPVas defined above. In certain embodiments, a nucleic acid of theinvention encodes a G protein, a N protein, a P protein, a M protein, aF protein, a M2-1 protein, a M2-2 protein, a SH protein, or a L proteinof subgroup A of a mammalian MPV as defined above. In certainembodiments, a nucleic acid of the invention encodes a G protein, a Nprotein, a P protein, a M protein, a F protein, a M2-1 protein, a M2-2protein, a SH protein, or a L protein of subgroup B of a mammalian MPVas defined above. In certain embodiments, a nucleic acid of theinvention encodes a G protein, a N protein, a P protein, a M protein, aF protein, a M2-1 protein, a M2-2 protein, a SH protein, or a L proteinof variant A1 of a mammalian MPV as defined above. In certainembodiments, a nucleic acid of the invention encodes a G protein, a Nprotein, a P protein, a M protein, a F protein, a M2-1 protein, a M2-2protein, a SH protein, or a L protein of variant A2 of a mammalian MPVas defined above. In certain embodiments, a nucleic acid of theinvention encodes a G protein, a N protein, a P protein, a M protein, aF protein, a M2-1 protein, a M2-2 protein, a SH protein, or a L proteinof variant B1 of a mammalian MPV as defined above. In certainembodiments, a nucleic acid of the invention encodes a G protein, a Nprotein, a P protein, a M protein, a F protein, a M2-1 protein, a M2-2protein, a SH protein, or a L protein of variant B2 of a mammalian MPVas defined above.

In certain embodiments, the invention provides a nucleotide sequencethat is at least 50%, at least 55%, at least 60%, at least 65%, at least70%, at least 75%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 98%, at least 99%, or at least 99.5%identical to the nucleotide sequence of SEQ ID NO:18, SEQ ID NO:19, SEQID NO:20, or SEQ ID NO:21. In certain embodiments, the nucleic acidsequence of the invention, is at least 50%, at least 55%, at least 60%,at least 65%, at least 70%, at least 75%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or atleast 99.5% identical to a fragment of the nucleotide sequence of SEQ IDNO:18, SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:21, wherein the fragmentis at least 25 nucleotides, at least 50 nucleotides, at least 75nucleotides, at least 100 nucleotides, at least 150 nucleotides, atleast 200 nucleotides, at least 250 nucleotides, at least 300nucleotides, at least 400 nucleotides, at least 500 nucleotides, atleast 750 nucleotides, at least 1,000 nucleotides, at least 1,250nucleotides, at least 1,500 nucleotides, at least 1,750 nucleotides, atleast 2,000 nucleotides, at least 2,00 nucleotides, at least 3,000nucleotides, at least 4,000 nucleotides, at least 5,000 nucleotides, atleast 7,500 nucleotides, at least 10,000 nucleotides, at least 12,500nucleotides, or at least 15,000 nucleotides in length. In a specificembodiment, the nucleic acid sequence of the invention is at least 50%,at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, at least 99%, or at least 99.5% or 100% identical to one ofthe nucleotide sequences of SEQ ID NOS:84-118; SEQ ID NOS:154-233; SEQID NOS:318-321; SEQ ID NOS:326-329; SEQ ID NOS:334-337; SEQ IDNOS:342-345; SEQ ID NOS:350-353; SEQ ID NOS:354-357; SEQ ID NOS:362-365;SEQ ID NOS:370-373; SEQ ID NOS:378-381; or SEQ ID NOS:386-389.

In specific embodiments of the invention, a nucleic acid sequence of theinvention is capable of hybridizing under low stringency, mediumstringency or high stringency conditions to one of the nucleic acidsequences of SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:21.In specific embodiments of the invention, a nucleic acid sequence of theinvention is capable of hybridizing under low stringency, mediumstringency or high stringency conditions to one of the nucleic acidsequences of SEQ ID NOS:84-118; SEQ ID NOS:154-233; SEQ ID NOS:318-321;SEQ ID NOS:326-329; SEQ ID NOS:334-337; SEQ ID NOS:342-345; SEQ IDNOS:350-353; SEQ ID NOS:354-357; SEQ ID NOS:362-365; SEQ ID NOS:370-373;SEQ ID NOS:378-381; or SEQ ID NOS:386-389. In certain embodiments, anucleic acid hybridizes over a length of at least 25 nucleotides, atleast 50 nucleotides, at least 75 nucleotides, at least 100 nucleotides,at least 150 nucleotides, at least 200 nucleotides, at least 250nucleotides, at least 300 nucleotides, at least 400 nucleotides, atleast 500 nucleotides, at least 750 nucleotides, at least 1,000nucleotides, at least 1,250 nucleotides, at least 1,500 nucleotides, atleast 1,750 nucleotides, at least 2,000 nucleotides, at least 2,00nucleotides, at least 3,000 nucleotides, at least 4,000 nucleotides, atleast 5,000 nucleotides, at least 7,500 nucleotides, at least 10,000nucleotides, at least 12,500 nucleotides, or at least 15,000 nucleotideswith the nucleotide sequence of SEQ ID NO:18, SEQ ID NO:19, SEQ IDNO:20, or SEQ ID NO:21.

The invention further provides antibodies and antigen-binding fragmentsthat bind specifically to a protein of a mammalian MPV. An antibody ofthe invention binds specifically to a G protein, a N protein, a Pprotein, a M protein, a F protein, a M2-1 protein, a M2-2 protein, a SHprotein, or a L protein of a mammalian MPV. In specific embodiments, theantibody is a human antibody or a humanized antibody. In certainembodiments, an antibody of the invention binds specifically to a Gprotein, a N protein, a P protein, a M protein, a F protein, a M2-1protein, a M2-2 protein, a SH protein, or a L protein of a virus ofsubgroup A of a mammalian MPV. In certain other embodiments, an antibodyof the invention binds specifically to a G protein, a N protein, a Pprotein, a M protein, a F protein, a M2-1 protein, a M2-2 protein, a SHprotein, or a L protein of a virus of subgroup B of a mammalian MPV. Incertain, more specific, embodiments, an antibody of the invention bindsspecifically to a G protein, a N protein, a P protein, a M protein, a Fprotein, a M2-1 protein, a M2-2 protein, a SH protein, or a L protein ofa virus of variant A1 of a mammalian MPV. In other embodiments, theantibody of the invention binds specifically to a G protein, a Nprotein, a P protein, a M protein, a F protein, a M2-1 protein, a M2-2protein, a SH protein, or a L protein of a virus of subgroup A2 of amammalian MPV. In certain embodiments, an antibody of the inventionbinds specifically to a G protein, a N protein, a P protein, a Mprotein, a F protein, a M2-1 protein, a M2-2 protein, a SH protein, or aL protein of a virus of subgroup B1 of a mammalian MPV. In certain otherembodiments, an antibody of the invention binds specifically to a Gprotein, a N protein, a P protein, a M protein, a F protein, a M2-1protein, a M2-2 protein, a SH protein, or a L protein of a virus ofsubgroup B2 of a mammalian MPV.

6. VIRUS ISOLATION AND CHARACTERIZATION 6.1 Example 1 SpecimenCollection, Virus Isolation, Virus Characterization

Samples of nasopharyngeal aspirates were obtained from hosts to assayfor the presence of viruses, and also to characterize those identified.Nasopharyngeal aspirates were collected from children suffering fromrespiratory tract infection (RTI). In order to determine the identity ofthe cause of illness, all nasopharyngeal aspirates were tested by directimmunofluorescence assays (DNF) (See method in Example 9), usingfluorescence labeled antibodies against influenza virus types A and B,hRSV, and human parainfluenza virus (hPIV) types 1, 2, and 3. Viruseswere also isolated from nasopharyngeal aspirates using rapid shell vialtechniques, (Rothbarth et. al., 1999, J. of Virol. Methods 78:163-169)on various cell lines, including VERO cells, tertiary cynomolgous monkeykidney (tMK) cells, human endothelial lung (HEL) cells and marbin dockkidney (MDCK) cells. Samples showing cytopathic effects (CPE) after twoto three passages, that were negative in DIF assays, were tested byindirect immunofluorescence assays (WA) (See method in Example 11),using virus specific antibodies against influenza virus types A, B andC, hRSV types A and B, measles virus, mumps virus, human parainfluenzavirus (hPIV) types 1 to 4, sendai virus, simian virus type 5, andNew-Castle disease virus. Although for many cases the etiological agentcould be identified, some specimens were negative for all of the virusestested.

These 28 unidentified virus isolates grew slowly in tMK cells, poorly inVERO cells and A549 cells and barely in MDCK or chicken embryonatedfibroblast cells. Most of the virus isolates induced CPE on tMK cells,between days ten and fourteen. This was somewhat later than the CPEcaused by other viruses such as hRSV or hPIV. The CPE were virtuallyindistinguishable from that caused by hRSV or hPIV in tMK or other cellcultures, and were characterized by syncytium formation. Some of theeffects observed on the cells included rapid internal disruption,followed by detachment of the cells from the monolayer.

The supernatants of infected tMK cells were used for Electron Microscopy(EM) analysis, and they revealed the presence of paramyxovirus-likevirus particles ranging from 150 to 600 nanometers in diameter, withshort envelope projections ranging from 13 to 17 nanometers. Consistentwith the biochemical properties of enveloped viruses such as theParamyxoviridae family of viruses, standard chloroform or ethertreatment (Osterhaus et. al., 1985, Arch. of Virol. 86:239-25) resultedin a greater than 10⁴ TCID₅₀ reduction in infectivity of tMK cells.Virus-infected tMK cell culture supernatants did not displayhemagglutinating activity with turkey, chicken and guinea pigerythrocytes. During culture, the virus replication appeared to betrypsin dependent. These combined virological data demonstrated that thenewly identified virus was a taxonomic member of the Paramyxoviridaefamily.

RNA from tMK cells infected with 15 of the unidentified virus isolateswas extracted for use in reverse transcription and polymerase chainreaction (RT-PCR) analyses, using primer-sets specific forParamyxovirinae (K. B. Chua et al., 2000, Science 288:1432-1435) suchas: hPIV 1-4, sendai virus, simian virus type 5, New-Castle diseasevirus, hRSV, morbilli, mumps, Nipah, Hendra, Tupaia and Mapuera viruses.RT-PCR assays were performed under conditions of low stringency in orderto detect potentially related viruses. RNA isolated from homologousvirus stocks was used as a control. Whereas the available controlsreacted positive with the respective virus-specific primers, the newlyidentified virus isolates did not react with any primer set, indicatingthe virus was not closely related to the viruses tested.

Two of the virus-infected tMK cell culture supernatants were used toinoculate guinea pigs and ferrets intranasally. Sera samples werecollected from these animals at day zero, two weeks, and three weekspost inoculation. The animals displayed no clinical symptoms, however,the seroconversion of all of the animals was detected and measured invirus neutralization (VN) (See method in Example 16) assays and indirectIFA against the homologous viruses. The sera did not react in indirectIFA with any of the known paramyxoviruses described above or withpneumovirus of mice (PVM). The so far unidentified virus isolates werescreened, using the guinea pig and ferret pre- and post-infection sera.Of these, 28 were clearly positive by indirect WA, with thepost-infection sera suggesting that, the thus far unidentified viralisolates, were closely related or identical.

In order further characterize the virus, the phenotypic effects of virusinfection on a cell line was examined. In short, tMK cells were culturedin 24 well plates containing glass slides (Costar, Cambridge, UK), withthe medium described below supplemented with 10% fetal bovine serum(BioWhittaker, Vervier, Belgium). Before inoculation, the plates werewashed with PBS and supplied with Eagle's MEM with Hanks' salt (ICN,Costa mesa, CA), of which 0.5 L was supplemented with 0.26 g of NaHCO₃,0.025 M Hepes (Biowhittaker), 2 mM L-glutamine (Biowhittaker), 100 unitspenicillin, 100 μg streptomycin (Biowhittaker), 0.5 g lactalbumin(Sigma-Aldrich, Zwijndrecht, The Netherlands), 1.0 g D-glucose (Merck,Amsterdam, The Netherlands), 5.0 g peptone (Oxoid, Haarlem, TheNetherlands) and 0.02% trypsin (Life Technologies, Bethesda, Md.). Theplates were inoculated with the supernatant of the nasopharyngealaspirate samples (0.2 ml per well in triplicate), followed bycentrifuging at 840×g for one hour. After inoculation, the plates wereincubated at 37° C. for a maximum of 14 days, and the medium was changedonce a week while cultures were checked daily for CPE. After 14 days,the cells were scraped from the second passage and incubated for 14days. This step was repeated for the third passage. The glass slideswere used to demonstrate the presence of the virus by indirect IFA asdescribed below.

CPE were generally observed after the third passage, between days 8 to14, depending on the isolate. The CPE were virtually indistinguishablefrom that caused by hRSV or hP1V in tMK or other cell cultures, exceptthat hRSV induces CPE at around day 4. CPE were characterized bysyncytia formation, after which the cells showed rapid internaldisruption, followed by detachment of the cells from the monolayer. Forsome isolates, CPE were difficult to observe, and WA was used to confirmthe presence of the virus in these cultures. The observation that theCPE were indistinguishable from those of other viruses indicated thatdiagnosis could not be made from a visual examination of clinicalsymptoms.

6.2 Example 2 Seroprevalence in the Human Population

To study the seroprevalence of this virus in the human population, serafrom humans in different age categories were analyzed by indirect IFAusing tMK cells infected with one of the unidentified virus isolates.Studies revealed that antibodies to the virus could be detected in 25%of the children between six and twelve months. Furthermore, by the ageof five, nearly 100% of the children were seropositive. In total, 56sera samples examined by indirect IFA and by VN assay. For 51 of thesamples or 91%, the results of the VN assay, i.e., a titer greater than8, coincided with the results obtained with indirect WA, i.e., a titergreater than 32. Four samples that were found to be positive by WA, werenegative by the VN assay, i.e., titer less than 8, whereas one serumsample was negative by WA, i.e., titer less than 32, and was positive bythe VN test, i.e., a titer of 16 (FIG. 2).

IFA conducted on 72 sera samples taken from humans in 1958, with agesranging from 8-99 years, revealed a 100% seroprevalence rate, indicatingthe virus has been circulating in the human population for more than 40years. In addition, a number of these sera samples were used in VNassays to confirm the IFA data (FIG. 2). The seroprevalence dataindicate that the virus has been a significant source of infection inthe human population for many years.

The repeated isolation of this virus from clinical samples from childrenwith severe RTI indicates that the clinical and economic impact of MPVmay be high. New diagnostic assays based on virus detection and serologywould yield a more detailed analysis of the incidence rate and also ofthe clinical and economical impact of this viral pathogen.

The slight differences between the IFA and VN results (5 samples) mayhave been due to the fact that in the IFA, only IgG serum antibodieswere detected, whereas the VN assay detects both classes and sub-classesof antibodies. Alternatively, differences may have been due to thedifferences in sensitivity between both assays. For IFA, a thresholdvalue of 16 was used, whereas for VN a value of 8 was used.

Differences between results in the IFA and VN assays may also indicatepossible differences between serotypes of this newly identified virus.Since MPV seems to be most closely related to APV, it was speculatedthat the human virus may have originated from birds. Analysis of serumsamples taken from humans in 1958 revealed that MPV has been widespreadin the human population for more than 40 years, indicating that atentative zoonosis event must have taken place long before 1958.

6.3 Example 3 Genomic Sequence of HMPV Isolate 00-1

In order to obtain sequence information for the unknown virus isolates,a random PCR amplification strategy known as RAP-PCR (Welsh et. al.,1992, NAR 20:4965-4970) (see Example 19). In short, tMK cells wereinfected with one of the virus isolates (isolate 00-1) as well as withhPIV-1 that served as a positive control. After both cultures displayedsimilar levels of CPE, virus in the culture supernatants was purified oncontinuous 20-60% sucrose gradients. The gradient fractions wereinspected for virus-like particles by EM, and RNA was isolated from thefraction that contained approximately 50% sucrose, in whichnucleocapsids were observed. Equivalent amounts of RNA isolated fromboth virus fractions were used for RAP-PCR, after which samples were runside by side on a 3% NuSieve agarose gel. Twenty differentiallydisplayed bands specific for the unidentified virus were subsequentlypurified from the gel, cloned in plasmid pCR2.1 (Invitrogen) andsequenced (See Example 20) with vector-specific primers. A search forhomologies against sequences in the Genbank database, using the BLASTprogram available through the National Library of Medicine, found that10 out of 20 fragments displayed resemblance to APV/TRTV sequences.

These 10 fragments were located in the genes coding for thenucleoprotein (N; fragment 1 and 2), the matrix protein (M; fragment 3),the fusion protein (F; fragment 4, 5, 6, 7) and the polymerase protein(L; fragment 8, 9, 10) (FIG. 3). PCR primers were designed to completethe sequence information for the 3′ end of the viral genome based on ourRAP PCR fragments as well as published leader and trailer sequences forthe Pneumovirinae (Randhawa, et. al., 1997, J. Virol. 71:9849-9854).Three fragments were amplified, of which fragment A spanned the extreme3′ end of the N open reading frame (ORF), fragment B spanned thephosphoprotein (F) ORF and fragment C closed the gap between the M and FORFs (FIG. 16). Sequence analyses of these three fragments revealed theabsence of NS1 and NS2 ORFs at the extreme 3′ end of the viral genomeand positioning of the F ORF immediately adjacent to the M ORF. Thisgenomic organization resembled that of the metapneumovirus APV, whichwas also consistent with the sequence homology. Relation betweendifferent viruses could be deduced by comparing the amino acid sequenceof FIGS. 4A-4E with the amino acid sequence of the respective N proteinsof other viruses. Overall the translated sequences for the N, P, M and FORFs showed an average of 30-33% homology with members of the genusPneumovirus and 66-68% with members of the genus Metapneumovirus. Forthe SH and G ORFs, no discernable homology was found with members ofeither genera. The amino acid homologies found for the amino acidsequence of the N ORF showed about 40% homology with hRSV and 88% withAPV-C, its closest relative genetically. The amino acid sequence for theP ORF showed about 25% homology with hRSV and about 66-68% with APV-C,the M ORF showed about 36-39% with hRSV and about 87-89% with APV-C, theF ORF showed about 40% homology with hRSV and about 81% with APV-C, theM2-1ORF showed about 34-36% homology with pneumoviruses and 84-86% withAPV-C, the M2-2 ORF showed 15-17% homology with pneumoviruses and 56%with APV-C and the fragments obtained from the L ORF showed an averageof 44% with pneumoviruses and 64% with APV-C.

Genetic analyses of the N, M, P and F genes revealed that MPV has highersequence homology to the recently proposed genus Metapneumovirinae ascompared to the genus Pneumovirinae and thus demonstrates a genomicorganization similar to and resembling that of APV/TRTV. In contrast tothe genomic organization of the RSVs (‘3-NS1-NS2-N-P-M-SH-G-F-M2-L-5’),metapneumoviruses lack NS1 and NS2 genes and also have a differentgenomic organization, specifically between the M and L(‘3-N-P-M-F-M2-5H-G-L-5’) genes. The lack of ORFs between the M and Fgenes in the virus isolates of the invention, the lack of NS1 and NS2adjacent to N, and the high amino acid sequence homology found withinAPV led to the proposed classification of MPV isolated from humans asthe first member of the metapneumovirus genus of mammals, and morespecifically of humans.

Phylogenetic analyses revealed that the nine MPV isolates, from whichsequence information was obtained, are closely related. Althoughsequence information was limited, they appeared to be more closelyrelated to one another than to any of the avian metapneumoviruses. Ofthe four serotypes of APV that have been described, serotype C appearedto be most closely related to MPV. This conclusion was based upon thenucleotide sequence similarities of the N, P, M and F genes. It shouldbe noted however, that for serotype D, only partial sequences of the Fgene were available from Genbank, and for serotype B, only M, N, and Fsequences were available. Our MPV isolates formed two clusters inphylogenetic trees. For both hRSV and APV, different genetic andserological subtypes have been described. Whether the two geneticclusters of MPV isolates represent serogical subgroups that are alsofunctionally different remains unknown at present. Our serologicalsurveys showed that MPV is a common human pathogen.

6.4 Example 4 Further Characterization of Associated Genes

Sequence analyses of the nucleoprotein (N), phosphoprotein (P),matrixprotein (M) and fusion protein (F) genes of MPV revealed thehighest degree of sequence homology with APV serotype C, the avianpneumovirus found primarily in birds in the United States. Theseanalyses also revealed the absence of non-structural proteins NS 1 andNS2 at the 3′ end of the viral genome and positioning of the fusionprotein immediately adjacent to the matrix protein. The sequences of the22K (M2) gene, the small hydrophobic (SH) gene, the attachment (G) gene,the polymerase (L) gene, the intergenic regions, and the trailersequences were determined. In combination with the sequences describedpreviously, the sequences presented here completed the genomic sequenceof MPV with the exception of the extreme 12-15 nucleotides of thegenomic termini and establish the genomic organization of MPV. Side byside comparisons of the sequences of the MPV genome with those of APVsubtype A, B and C, RSV subtype A and B, PVM and other paramyxovirusesprovides strong evidence for the classification of MPV in theMetapneumovirus genus.

GENE ENCODING THE NUCLEOPROTEIN (N): As shown above, the first gene inthe genomic map of MPV codes for a 394 amino acid (aa) protein and showsextensive homology with the N protein of other pneumoviruses. The lengthof the N ORF is identical to the length of the N ORF of APV-C (Table 5)and is smaller than those of other paramyxoviruses (Barr et al., 1991, JGen Virol 72:677-85). Analysis of the amino acid sequence revealed thehighest homology with APV-C (88%), and only 7-11% with otherparamyxoviruses (Table 6).

Three regions of similarity between viruses belonging to the orderMononegavirales were identified: A, B and C (FIG. 22) (Barr et al.,1991, J. Gen. Virol. 72: 677-85). Although similarities are highestwithin a virus family, these regions are highly conserved between virusfamilies observed. In all three regions MPV revealed 97% aa sequenceidentity with APV-C, 89% with APV-B, 92% with APV-A, and 66-73% with RSVand PYM. The region between aa residues 160 and 340 appears to be highlyconserved among metapneumoviruses and to a somewhat lesser extent thePneumovirinae (Miyahara et al., 1991, Arch. Viral. 124:255-68; L₁ etal., 1996, Virus Res. 41:185-91; Ban, 1991, J. Gen. Virol. 72:677-85).

GENE ENCODING THE PHOSPHOPROTEIN (P): The second ORF in the genome mapcodes for a 294 aa protein which shares 68% aa sequence homology withthe P protein of APV-C, and only 22-26% with the P protein of RSV (Table7). The P gene of MPV contains one substantial ORF and in that respectis similar to P from many other paramyxoviruses (Reviewed in Lamb et.al., Fields virology, (B. N. Knipe, P. M. Hawley, ed., LippencottRaven),Philadelphia, 1996; Sedlmeier et al., 1998, Adv. Virus Res. 50:101-39).

In contrast to APV A and B and PVM and similar to RSV and APV-C the MPVP ORF lacks cysteine residues. A region of high similarity between allpneumoviruses (amino acids 185-241) plays a role in either the RNAsynthesis process or in maintaining the structural integrity of thenucleocapsid complex (Ling et al., 1995, Virus Res. 36:247-57). Thisregion of high similarity is also found in MPV (FIG. 6) especially whenconservative substitutions are taken into account, showing 100%similarity with APYC, 93% with APV-A and B, and approximately 81% withRSV. The C-terminus of the MPV P protein is rich in glutamate residuesas has been described for APVs (Ling, et al., 1995, Virus Res.36:247-57).

GENE ENCODING THE MATRIX (M) PROTEIN: The third ORF of the MPV genomeencodes a 254 aa protein, which resembles the M ORFs of otherpneumoviruses. The M ORF of MPV has exactly the same size as the M ORFsof other metapneumoviruses and shows high aa sequence homology with thematrix proteins of APV (78-87%), lower homology with those of iRSV andPVM (37-38%), and 10% or less homology with those of otherparamyxoviruses (Table 6).

The sequences of matrix proteins of all pneumoviruses were compared anda conserved heptadpeptide at residue 14 to 19 was found to alsoconserved in MPV (FIG. 7) (Easton et al. 1997, Virus Res. 48:27-33). ForRSV, PVM and APV, small secondary ORFs within or overlapping with themajor ORF of M have been identified (52 aa and 51 aa in bRSV, 75 aa inRSV, 46 aa in PVM and 51 aa in APV) (Yu et al., 1992, Virology186:426-34; Easton et al., 1997, Virus Res. 48:27-33; Samal et al.,1991, J. Gen. Virol. 72:715-20; Satake et al., 1995, J. Virol. 50:92-9).One small ORF of 54 aa residues was found within the major M ORF(fragment 1, FIG. 8), starting at nucleotide 2281 and one small ORF of33 aa residues was found overlapping with the major ORF of M starting atnucleotide 2893 (fragment 2, FIG. 8). Similar to the secondary ORFs ofRSV and APV there is no significant homology between these secondaryORFs and secondary ORFs of the other pneumoviruses, and apparent startor stop signals are lacking. Furthermore, there have not been any reportof protein synthesis occurring from these secondary ORFs.

GENE ENCODING THE FUSION PROTEIN: The F ORF of MPV is located adjacentto the M ORF, a feature that is characteristic of members of theMetapneumovirus genus. The F gene of MPV encodes a 539 aa protein, whichis two aa residues longer than F of APV-C. Analysis of the aa sequencerevealed 81% homology with APV-C, 67% with APV-A and B, 33-39% withpneumovirus F proteins and only 10-18% with other paramyxoviruses (Table6). One of the conserved features among F proteins of paramyxoviruses,and also seen in MPV is the distribution of cysteine residues (Morrisonet al., 1988, Virus Res. 10:113-35; Yu et al., 1991, J. Gen. Virol.72:75-81). The metapneumoviruses share 12 cysteine residues in E1 (7 areconserved among all paramyxoviruses), and two in E2 (1 is conservedamong all paramyxoviruses). Of the 3 potential N-linked glycosylationsites present in the F ORF of MPV, none are shared with RSV and two(position 74 and 389) are shared with APV. The third, unique, potentialN-linked glycosylation site for MPV is located at position 206 (FIG. 9).

Despite the low sequence homology with other paramyxoviruses, the Fprotein of MPV revealed typical fusion protein characteristicsconsistent with those described for the F proteins of otherParamyxoviridae family members (Morrison et. al., 1988, Virus Res.10:113-35). F proteins of Paramyxoviridae members are synthesized asinactive precursors (F0) that are cleaved by host cell proteases whichgenerate amino terminal E2 subunits and large carboxy terminal F1subunits. The proposed cleavage site (Collins et al., Fields virology(B. N. Knipe, P. M. Howley, ed., Lippencott-Raven), Philadelphia, 1996)is conserved among all members of the Paramyxoviridae family. Thecleavage site of MPV contains the residues RQSR. Both arginine (R)residues are shared with APV and RSV, but the glutamine (Q) and serine(S) residues are shared with other paramyxoviruses such as humanparainfluenza virus type 1, Sendai virus and morbilliviruses.

The hydrophobic region at the amino terminus of F1 is thought tofunction as the membrane fusion domain and shows high sequencesimilarity among paramyxoviruses and morbilliviruses and to a lesserextent the pneumoviruses (Morrison et al., 1988, Virus Res. 10:113-35).These 26 residues (position 137-163, FIG. 9) are conserved between MPVand APV-C, which is in agreement with this region being highly conservedamong the metapneumoviruses (Naylor et al., 1998, J. Gen. Virol.79:1393-1398; Seal et al., 2000, Virus Res. 66:139-47).

As is seen for the F2 subunits of APV and other paramyxoviruses, MPVrevealed a deletion of 22 aa residues compared with RSV (position107-128, FIG. 9). Furthermore, for RSV and APV, the signal peptide andanchor domain were found to be conserved within subtypes and displayedhigh variability between subtypes (Plows et al., 1995, Virus Genes11:37-45; Naylor et al., 1998, J. Gen. Virol. 79:1393-1398). The signalpeptide of MPV (aa 10-35, FIG. 9) at the amino terminus of F2 exhibitssome sequence similarity with APV-C (18 out of 26 aa residues aresimilar), and less conservation with other APVs or RSV. Much morevariability between subtypes is seen in the membrane anchor domain atthe carboxy terminus of E1, although some homology is still seen withAPV-C.

GENE ENCODING THE M2 PROTEIN: The M2 gene is unique to the Pneumovirinaeand two overlapping ORFs have been observed in all pneumoviruses. Thefirst major ORF represents the M2-1 protein which enhances theprocessivity of the viral polymerase (Collins et al., 1995, Proc. Natl.Acad. Sci. USA 92:11563-7; Collins et. al., Fields virology (B. N.Knipe, P. M. Howley, ed., Lippencott-Raven), Philadelphia, 1996) and itsread through of intergenic regions (Hardy et al., 1998, J. Viral.72:520-6; Fearns et al., 1999, J. Virol. 73:5852-64). The M2-1 gene forMPV, located adjacent to the F gene, encodes a 187 aa protein, andreveals the highest (84%) homology with M2-1 of APV-C. Comparison of allpneumovirus M2-1 proteins revealed the highest conservation in theamino-terminal half of the protein (Collins et al., 1990, J. Gen. Virol.71:3015-20; Zamora et al., 1992, J. Gen. Virol. 73:737-41; Ahmadian etal., 1999, J. Gen. Virol. 80:2011-6), which is in agreement with theobservation that MPV displays 100% similarity with APV-C in the first 80aa residues of the protein (FIG. 10). The MPV M2-1 protein contains 3cysteine residues located within the first 30 aa residues that areconserved among all pneumoviruses. Such a concentration of cysteines isfrequently found in zinc-binding proteins (Cuesta et al., 2000, Gen.Virol. 74, 9858-67).

The secondary ORFs (M2-2) that overlap with the M2-1 ORFs ofpneumoviruses are conserved in location but not in sequence and arethought to be involved in the control of the switch between virus RNAreplication and transcription (Collins et al., 1985, J. Viral. 54:65-71;Elango et al., 1985, J. Viral. 55:101-10; Baybutt et. al., 1987, J. Gen.Virol. 68:2789-96; Collins et al., 1990, J. Gen. Virol. 71:3015-20; Linget al., 1992, J. Gen. Viral. 73:1709-15; Zamora et al., 1992, J. Gen.Virol. 73:737-41; Alansari et al., 1994, J. Gen. Virol: 75:401-404;Ahmadian et al., 1999, J. Gen. Virol. 80: 2011-6). For MPV, the M2-2 ORFstarts at nucleotide 512 in the M2-1 ORF (FIG. 8), which is exactly thesame start position as for APV-C. The length of the M2-2 ORFs are thesame for APV-C and MPV, 71 aa residues. Sequence comparison of the M2-2ORF (FIG. 10) revealed 64% aa sequence homology between MPV and APV-Cand only 44-48% aa sequence homology between MPV and APV-A and B.

SMALL HYROPHOBIC (SH) GENE ORF: The gene located adjacent to M2 of hMPVprobably encodes a 183 aa SH protein (FIG. 8). There is no discerniblesequence identity between this ORF and other RNA virus genes or geneproducts. This is not surprising since sequence similarity betweenpneumovirus SH proteins is generally low. The aa composition of the SHORF is relatively similar to that of APV, RSV and PVM, with a highpercentage of threonine and serine residues (22%, 18%, 19%, 20.0%, 21%and 28% for hMPV, APV, RSV A, RSV B, bRSV and PVM respectively). The SHORF of hMPV contains 10 cysteine residues, whereas APV SH contains 16cysteine residues. The SH ORF of hMPV contains two potential N-linkedglycosylation sites (aa 76 and 121), whereas APV has one, RSV has two orthree and PVM has four.

The hydrophilicity profiles for the putative hMPV SH protein and SH ofAPV and RSV revealed similar characteristics (FIG. 11B). The SH ORFs ofAPV and hMPV have a hydrophilic N-terminus, a central hydrophobic domainwhich can serve as a potential membrane spanning domain (aa 30-53 forhMPV), a second hydrophobic domain (aa 155-170) and a hydrophilicC-terminus. In contrast, RSV SH appears to lack the C-terminal part ofthe APV and hMPV ORFs. In all pneumovirus SH proteins the hydrophobicdomain is flanked by basic aa residues, which are also found in the SHORF for hMPV (aa 29 and 54).

GENE ENCODING THE ATTACHMENT GLYCOPROTEIN (G): The putative G ORF ofhMPV is located adjacent to the putative SH gene and encodes a 236 asprotein (nt 6262-6972, FIG. 8). A secondary small ORF is foundimmediately following this ORF, potentially coding for 68 aa residues(nt 6973-7179) but lacking a start codon. A third potential ORF in thesecond reading frame of 194 aa residues is overlapping with both ofthese ORFs but also lacks a start codon (nt 6416-7000). This ORF isfollowed by a potential fourth ORF of 65 aa residues in the same readingframe (nt 7001-7198), again lacking a start codon. Finally, a potentialORF of 97 aa residues (but lacking a start codon) is found in the thirdreading frame (nt 6444-6737, FIG. 8). Unlike the first ORF, the otherORFS do not have apparent gene start or gene end sequences (see below).Although the 236 aa G ORE probably represents at least a part of thehMPV attachment protein it cannot be excluded that the additional codingsequences are expressed as separate proteins or as part of theattachment protein through some RNA editing event. It should be notedthat for APV and RSV no secondary ORFs after the primary G ORF have beenidentified but that both APV and RSV have secondary ORFs within themajor ORF of G. However, evidence for expression of these ORFs islacking and there is no sequence identity between the predicted aasequences for different viruses (Ling et al., 1992, J. Gen. Virol.73:1709-15). The secondary ORFs in hMPV G do not reveal characteristicsof other G proteins and whether the additional ORFs are expressedrequires further investigation.

BLAST analyses with all ORFs revealed no discernible sequence identityat the nucleotide or aa sequence level with other known virus genes orgene products. This is in agreement with the low percentage sequenceidentity found for other G proteins such as those of hRSV A and B (53%)(Johnson et al., 1987, J. Virol. 61:163-6) and APV A and B (38%) (Juhaszand Easton, 1994, J. Gen. Virol. 75:2873-80).

Whereas most of the hMPV ORFs resemble those of APV both in length andsequence, the putative G ORF of 236 aa residues of hMPV is considerablysmaller than the G ORF of APV (Table 4). The aa sequence revealed aserine and threonine content of 34%, which is even higher than the 32%for RSV and 24% for APV. The putative G ORF also contains 8.5% prolineresidues, which is higher than the 8% for RSV and 7% for APV. Theunusual abundance of proline residues in the G proteins of APV, RSV andhMPV has also been observed in glycoproteins where it is a majordeterminant of the proteins three dimensional structure (Collins andWertz, 1983, PNAS 80:3208-12; Wertz et al., 1985, PNAS 82:4075-9;Jentoft, 1990, Trends Biochem. Sci. 15:291-4). The G ORF of hMPVcontains five potential N-linked glycosylation sites, whereas hRSV hasseven, bRSV has five and APV has three to five.

The predicted hydrophilicity profile of hMPV G revealed characteristicssimilar to the other pneumoviruses. The N-terminus contains ahydrophilic region followed by a short hydrophobic area (aa 33-53 forhMPV) and a mainly hydrophilic C-terminus (FIG. 12B). This overallorganization corresponds well with regions in the G protein of APV andRSV. The putative G ORF of hMPV contains only 1 cysteine residue incontrast to RSV and APV (5 and 20 respectively). Of note, only two ofthe four secondary ORFs in the G gene contained one additional cysteineresidue and these four potential ORFs revealed 12-20% serine andthreonine residues and 6-11% proline residues.

POLYMERASE GENE (L): In analogy to other negative strand viruses, thelast ORF of the MPV genome is the RNA-dependent RNA polymerase componentof the replication and transcription complexes. The L gene of MPVencodes a 2005 aa protein, which is one residue longer than the APV-Aprotein (Table 5). The L protein of MPV shares 64% homology with APV-A,42-44% with RSV, and approximately 13% with other paramyxoviruses (Table6). Six conserved domains within the L proteins of non-segmentednegative strand RNA viruses were identified; it was found that thedomain three contained the four core polymerase motifs that are thoughtto be essential for polymerase function (Poch et al., 1990, J. Gen.Virol. 71:1153-62; Poch et al., 1989, EMBO J. 8:3867-74). These motifs(A, B, C and D) are well conserved in the MPV L protein: in motifs A, Band C: MPV shares 100% similarity with all pneumoviruses and in motif DMPV shares 100% similarity with APV and 92% with RSVs. For all of domainIII (aa 627-903 in the L ORF), MPV shares 77% identity with APV, 61-62%with RSV and 23-27% with other paramyxoviruses (FIG. 13). In addition tothe polymerase motifs the pneumovirus L proteins contain a sequencewhich conforms to a consensus ATP binding motif K(X)₂₁ GEGAGN(X)₂₀K(Stec et al., 1991, Virology 183:273-87). The MPV L ORF contains asimilar motif as APV, in which the spacing of the intermediate residuesis shifted by one residue: K(X)₂₂ GEGAGN(X)₁₉K.

TABLE 5 LENGTHS OF THE ORFs OF MPV AND OTHER PARAMYXOVIRUSES N¹ P M FM2-1 M2-2 SH G L MPV 394 294 254 539 187 71 183  236 2005 APV A 391 278254 538 186 73 174  391 2004 APV B 391 279 254 538 186 73 ** 414 ** APVC 394 294 254 537 184 71 ** ** ** APV D ** ** ** ** ** ** ** 389 ** hRSVA 391 241 256 574 194 90 64 298 2165 hRSV B 391 241 249 574 195 93 65299 2166 bRSV 391 241 256 569 186 93 81 257 2162 PVM 393 295 257 537 17677 92 396 ** others³ 418-542 225-709 335-393 539-565 **** **** **** ****2183-2262 Legend for Table 5: * = length in amino acid residues, ** =sequences not available, *** = others: human parainfluenza virus type 2and 3, Sendai virus, measles virus, nipah virus, phocine distempervirus, and New Castle Disease virus, **** = ORF not present in viralgenome.

TABLE 6 AMINO ACID SEQUENCE IDENTITY BETWEEN THE ORFs OF MPV AND THOSEOF OTHER PARAMYXOVIRUSES N P M F M2-1 M2-2 L APV A 69 55 78 67 72 26 64APV B 69 51 76 67 71 27 ** APV C 88 68 87 81 84 56 ** hRSV A 42 24 38 3436 18 42 hRSV B 41 23 37 33 35 19 44 bRSV 42 22 38 34 35 13 44 PVM 45 2637 39 33 12 ** others³ 7-11 4-9 7-10 10-18 **** **** 13-14 Legend forTable 6: * = No sequence homologies were found with known G and SHproteins and were thus excluded, ** = Sequences not available, *** = Seelist in Table 4, denoted by same (***), **** = ORF absent in viralgenome.

6.5 Example 5 Genomic Sequencing of HMPV Isolate 1-99

Another isolate of hMPV (1-99) was also identified and sequenced. Inorder to do so, the hMPV isolate 1-99 was propagated on tertiary monkeykidney cells exactly as described before (van den Hoogen et al., 2001,Nature Medicine 7(6):719-724). Viral RNA was isolated using theMagnaPure LC isolation system (Roche Applied Science) and the totalnucleic acid kit protocol. RNA was converted into cDNA using standardprotocols, with random hexamers (Progema Inc. Leiden) as primers. ThiscDNA was kept at −20° C. or lower until used for sequence analysis.Primers used throughout this project were based on the sequencesavailable from the prototype hMPV 1-00 strain, or obtained aftersequence analysis using the hMPV strain 1-99.

PCR fragments were made ranging in size up to 1600 base-pairs togenerate overlapping fragments. Sequence analysis was performed on thePCR fragments using standard technology and an ABI 3100 capillarysequence instrument (Applied Biosystems, Nieuwerkerk Issel). Thenucleotide sequences generated were compared initially with theprototype hMPV strain 1-00 for comparison. Blast software was used forcomparison with related sequences in the GenBank database. For furtheranalysis of the sequences, DNASTAR software was used (DNASTAR Inc,Madison Wis., U.S.A.) and for phylogenetic analysis, the ClustalWsoftware program was used.

Initially, sequences for the 1-99 isolate were obtained using primersthat were designed based on sequence information from the 1-00 isolate.However, since some parts of the genome could not be sequenced based onthe information from the 1-00 isolate, new primers based on sequenceinformation from the 1-99 isolate, as well from information madeavailable through the sequencing of the 3′ and 5′ end of the 1-00isolate, were used.

The prototype sequence of the hMPV isolate 1-99 contained 13,223base-pairs, sequenced in a total of 227 individual sequences, with anaverage length of 404 base-pairs. The sequence is SEQ ID NO:18.

The length of the open reading frames of hMPV 1-99 and otherParamyxoviruses, both in absolute size and percentage amino acididentity are shown in Table 7. Most identity between the 1-99 and 1-00strains was observed in the genes coding for N protein (95.2%), M(97.3%), F (93.7%), L (94.1%) and M2-1 (94.1%) with percentages homologyof over 90%. The homology of the P and M2-2 genes between both strainswas found to be 86.1 and 88.9% respectively. Also, the isolate is mostlyrelated to the subtype C of the avian Metapneumovirus, with amino acididentities in the N protein (88.6%), M protein (87.1%) and M2-1 protein(84.3%). The identity with the P and M2-2 proteins is lower at 67.8% and56.9% respectively.

The genes of the prototype 1-00 and 1-99 strains are identical on thegenomic map, with the same number of amino acids for N, P, M, F, M21 andM2-2 protein. The putative SH gene is 6 amino acids shorter, the Gprotein is 12 amino acids shorter, and the L gene of the 1-00 and 1-99strain are the same size.

Finally, the start of the genes on the genomic map and the non-codingsequences located between the genes, have been summarized in Table 8.

In summary, the sequence information of the 1-99 strain of the humanMetapneumovirus clearly demonstrates the genetic relation of 1-99 withthe prototype strain 1-00, sharing identical genomic map organization.Less phylogenetic relation is observed with the subtype C of APV.

TABLE 7 N P M F M21 M22 SH G L LENGTH OF THE ORFS OF HMPV 1-99 AND OTHERPARAMYXOVIRUSES (NO. OF AMINO ACID RESIDUES) 1-99 394 294 254 539 187 71177 224 1937 1-00 394 294 254 539 187 71 183 236 2005 APV-A 391 278 254538 186 73 174 391 2004 APV-B 391 279 254 538 186 73 414 APV-C 394 294254 537 184 71 hRSV-A 391 241 256 574 194 90 64 298 2165 hRSV-B 391 241256 574 195 90 65 299 2166 bRSV 391 241 256 574 186 90 81 257 2162 PVM393 295 257 537 176 98 92 396 PERCENTAGE OF THE AMINO ACID SEQUENCEIDENTITY BETWEEN HMPV 1-99 AND OTHER PARAMYXOVIRUSES 1-00 95.2 86.1 97.393.7 94.1 88.9 59 32.4 94.1 APV-A 68.9 58.1 76.1 67.5 69 25 13.1 14.263.7 APV-B 69.1 53.9 76.5 66.8 65.8 26.4 APV-C 88.6 67.8 87.1 80.5 84.356.9 bRSV 41.1 28.1 36.9 35 32.6 9.7 12.2 15.6 46.5 hRSV-A 41.1 26 37.632.2 35.6 6.2 16 46.9 hRSV-B 40.6 26 36.9 34.4 34 13.9 21.2 15.6 47 PVM43.7 22.4 39.2 38.8 5.4 8

TABLE 8 SUMMARY OF GENE START SEQUENCES ON THE GENOMIC MAP AND THENON-CODING SEQUENCES LOCATED BETWEEN THE GENES. Pos. ORF StopNon-coding sequence Gene Start Start Pos. ORF 1 LeACGAGAAAAAAACGCGUAUAAAUU GGGACAAAUAAAA AUG    54 N AAAUUCCAAACAAAAC 1238N UAA UUAAAAAACU GGGACAAGUCAAA AUG  1262  P 2146 P UAGUUUAAUAAAAAUAAACAAU GGGACAAGUCAAG AUG  2179  M 2943 M UAAAAAUAACUGUCUUAAUCAAUAAUU GGGACAAAUAAAA AUG  3065  FGCUUAUAUAACUCUAGAGAUUAAU AAGCUUAUUAUUAUAGUUAUAUAAAAAUAAAUUAGAAUUAGAAGGGCA UCAAUAGAAAGC 4684 F UAG UUAAUUAAAAAAUGGGACAAAUCAUC AUG  4711  M2 5437 M2 UAG UAAAAAAUAAAAAUAGAAUGGGAUAAAUGACA AUG  5470  SH 6003 SH UAA AAUAACACGGSUUUSAACAUUAAAGGGACAAGUGGCU AUG  6210  G AUSAGAACAACCUCCACCCAGGUCUAUCAAUACAGUGGUUUAGCCAUU UAAAAACCGAAUAUUAUCUAGGCUGCACGACACUUUGCAAUAAUAUGC AAUAGUCAAUAGUUAAACCACUGCUGCAAACUCAUCCAUAAUAUAAUC ACUGAGUAAUACAAAACAAGAAAA U 6884 G UAGAGAGGUGCAAAACUCAAAUGAGCA GGGAUAAAUGACA AUG  7124  LCAACACACAAACAUYCCAUCCAAG UAGUUAACAAAAAACCACAAAAUAACCUUGAAAACCAAAAAACCAAAA CAUAAACCCAGACCCAGAAAAACAUAGACACCAUAUGGAAGGUUCUAG CAUAUGCACCAAUGAGAUGGCAUCUGUUCAUGUAUCAAUAGCACCACC AUCAUUCAAGGAAUAAGAAGAGGC GAAAAUUUAA 13009 L UGAAUUAAACUAUGAUUUCUUUGAAGC AUG 13243 Tr AUUAGAGAACACAUACCCCAAUAUGAUCAAGCUUAUAGAUAAUUUGGG AAAUGCAGAAAUAAAGAAACUAAUCMAGGUCMCUGGGUAUAUGCUUGU GAGUAAGAAGUAAUAAUAAUGAUAAUGAUUAACCAUAAUCUCMCMCMA CUGAGAAAAUAAUCGUCUAACAGUUUAGUUGAUCAUUAGUUAUUUAAA AUUAUAAAAUAGUAACUA

6.6 Example 6 Phylogenetic Relationships

Phylogenetic approaches can be used in order to identify therelationships among groups of viruses, i.e. between MPV and otherviruses. Additionally, phylogenetic relationships can be determined fordifferent isolates of the same type of virus. Phylogenetic trees weredetermined to determine relationships between MPV and other viruses, andalso to determine relationships between the different isolates of hMPV.For example, phylogenetic trees can be generated, using nucleotide orprotein sequence data, in order to illustrate the relationship betweenMPV and different viruses. Alternatively, phylogenetic trees can begenerated, using nucleotide or protein sequence data, in order toillustrate the relationship between various isolates of hMPV.

PHYLOGENETIC RELATIONSHIPS BETWEEN HMPV AND DIFFERENT VIRUSES: AlthoughBLAST searches using nucleotide sequences obtained from the unidentifiedvirus isolates revealed homologies primarily with members ofPneumovirinae, homologies that were based on protein sequences revealedsome resemblance with other paramyxoviruses as well. As an indication ofthe relationship between the newly identified virus isolates and membersof Pneumovirinae, phylogenetic trees were constructed based on the N, P,M and F ORFs of these viruses. In all four phylogenetic trees, the newlyidentified virus isolate was most closely related to APV (FIG. 14). Fromthe four serotypes of APV that have been described (Bayon-Auboyer etal., 2000, J. Gen. Virol 81:2723-2733), APV serotype C, themetapneumovirus found primarily in birds in the USA, showed the closestresemblance to the newly identified virus. It should be noted however,that only partial sequence information for APV serotype D is available.

For all phylogenetic trees, DNA sequences were aligned using theClustalW software package and maximum likelihood trees were generatedusing the DNA-ML software package of the Phylip 3.5 program using 50 or100 bootstraps and 3 jumbles (Brandenburg et al., 1997, J. Med. Virol.52:97-104). Previously published sequences that were used for thegeneration of phylogenetic trees are available from Genbank underaccessions numbers: For all ORFs: hRSV: NC001781; bRSV: NC001989; Forthe F ORF: PYM, D11128; MV-A, D00850; MV-B, Y14292; MV-C, AF187152; Forthe N ORF: PVM, D10331; MV-A, U39295; MV-B, U39296; MV-C, M176590; Forthe M ORF: PMV, U66893; MV-A, X58639; MV-B, U37586; MV-C, AE262571; Forthe P ORF: PVM, 09649; MV-A, U22110, MV-C, AF176591.

As an indicator of the relationship between MPV and members of thePneumovirinae, phylogenetic trees based on the N, P, M, and F ORFs wereconstructed previously (van den Hoogen et al., 2001, Nat. Med.7(6):19-24) and revealed a close relationship between MPV and APV-C.Because of the low homology of the MPV SH and G genes with those genesof other paramyxoviruses, reliable phylogenetic trees for these genescannot be constructed. In addition, the distinct genomic organizationbetween members of the Pneumovirus and Metapneumovirus genera make itimpossible to generate phylogenetic trees based on the entire genomicsequence. Trees for the M2 and L genes were constructed in addition tothose previously published. Both these trees confirmed the closerelation between APV and MPV within the Pneumovirinae subfamily (FIG.15).

To construct phylogenetic trees, DNA sequences were aligned using theClustalW software package and maximum likelihood trees were generatedusing the DNA-ML software package of the Phylip 3.5 program using 100bootstraps and 3 jumbles. Bootstrap values were computed for consensustrees created with the PHYLIP consensus package.

Based upon phylogenetic analyses of the different isolates of hMPVobtained so far, two major genotypes have been identified with virusisolate 00-1 being the prototype of genotype A and isolate 99-1 theprototype of genotype B.

It is hypothesized that the genotypes are related to subtypes and thatre-infection with viruses from both subgroups occur in the presence ofpre-existing immunity and the antigenic variation may not be strictlyrequired to allow re-infection. Furthermore, hMPV appears to be closelyrelated to avian pneumovirus, a virus primarily found in poultry. Thenucleotide sequences of both viruses show high percentages of homology,with the exception of the SH and G proteins. The viruses appear tocross-react in tests that are based primarily on the nucleoprotein andmatrixprotein, however, they respond differently in tests that are basedon the attachment proteins. The differences in virus neutralizationtiter provide further proof that the two genotypes of hMPV are twodifferent serotypes of one virus, where APV is a different virus.

PHYLOGENETIC RELATIONSHIPS BETWEEN DIFFERENT HMPV ISOLATES: Phylogeneticapproaches can also be used in order to identify the relationships amongdifferent isolates of MPV. For example, phylogenetic trees can begenerated, using nucleotide or protein sequence data of MPV, in order toillustrate the relationship between a number of MPV isolates that areobtained from different subjects. This approach is useful inunderstanding the differences that occur within the population of MPVviruses.

To determine the relationship of our various newly identified virusisolates, phylogenetic trees were constructed based on sequenceinformation obtained from eight to nine isolates (8 for F, 9 for N, Mand L). RT-PCR was used with primers designed to amplify short fragmentsin the N, M, F, P, SH and L ORFs, that were subsequently sequenceddirectly. The nine virus isolates that were previously found to berelated in serological terms (see above) were also found to be closelyrelated genetically. In fact, all nine isolates were more closelyrelated to one another than to APV. Although the sequence informationused for these phylogenetic trees was limited, it appears that the nineisolates can be divided in two groups, with isolate 94-1, 99-1 and 99-2clustering in one group and the other six isolates (94-2; 93-1; 93-2;93-3; 93-4; 00-1) in the other (FIG. 16).

An alignment of the F genes of different isolates of hMPV of all fourvariants, variant A1, A2, B1, or B2, is shown in FIGS. 17A-17K.

An alignment of the F proteins of different isolates of hMPV of all fourvariants, variant A1, A2, B1, or B2, is shown in FIGS. 18A-18D.

An alignment of the G genes of different isolates of hMPV of all fourvariants, variant A1, A2, B1, or B2, is shown in FIGS. 19A-19J.

An alignment of the G proteins of different isolates of hMPV of all fourvariants, variant A1, A2, B1, or B2, is shown in FIGS. 20A-20C.

A phylogenetic tree based on the F gene sequences showing thephylogenetic relationship of the different hMPV isolates and theirassociation with the respective variants of hMPV is shown in FIG. 21.Further, a phylogenetic tree based on the G gene sequences showing thephylogenic relationship of the different hMPV isolates and theirassociation with the respective variants of hMPV is shown in FIG. 22.The phylogenetic trees were calculated using DNA maximum likelihood with50 bootstraps and 3 jumbles.

Sequence identities between different genes of hMPV isolate 00-1 withdifferent genes of hMPV isolate 99-1, APV serotype C, and APV serotype Aare listed in Table 9.

TABLE 9 ORF SEQUENCE IDENTITY BETWEEN HMPV ISOLATE 00-1 AND OTHERVIRUSES N P M F M2.1 M2.2 SH G L hMPV isolate 99-1 95 86 98 94 95 90 5733 94 APV serotype C 88 68 87 81 84 56 N.A. N.A. N.A. APV serotype A 6955 78 68 72 25 18  9 64

Originally, phylogenetic relationships were inferred for only ninedifferent isolates. Two potential genetic clusters were identified byanalyses of partial nucleotide sequences in the N, M, F and L ORFs ofvirus isolates. Nucleotide identity of 90-100% was observed within acluster, and 81-88% identity was observed between the clusters. Sequenceinformation obtained on more virus isolates confirmed the existence oftwo genotypes. Virus isolate 00-1, as a prototype of cluster A, andvirus isolate 99-1 as a prototype of cluster B, have been used in crossneutralization assays to test whether the genotypes are related todifferent serotypes or subgroups.

Using RT-PCR assays with primers located in the polymerase gene, thirtyadditional virus isolates were identified from nasopharyngeal aspiratesamples. Sequence information of parts of the matrix and polymerasegenes of these new isolates together with those of the previous nineisolates were used to construct phylogenetic trees (FIG. 15). Analysesof these trees confirmed the presence of two genetic clusters, withvirus isolate 00-1, as the prototype virus in group A and virus isolate99-1 as the prototype virus in group B. The nucleotide sequence identitywithin a group was more than 92%, while between the clusters theidentity was 81-85%.

6.7 Example 7 Leader Sequences of Human Metapneumovirus (HMPV) NL/1/00Genomic RNA

While the majority of genomic composition was determined, the authenticterminal sequences at the extreme ends were lacking. Using ligation ofthe viral RNA and subsequent PCR amplification of the ligated junctionand a combination of polyadenylation and 3′ RACE methods, the authenticnucleotide sequences were determined (FIG. 54). The sequence analysis ofPCR fragments generated by ligation of viral RNA ends revealed theLeader and Trailer sequences displayed in FIG. 26 (see, SEQ IDs 18-21).The trailer sequences obtained this way were consistent with thesequences expected from the trailer sequences of other paramyxoviruses,including APV. However, the leader sequence of only 2 out of 71 clonessequenced, contained AC as the terminal nucleotide residues that arefound in all paramyxoviruses to date. Therefore, the terminal nucleotidesequences of the hMPV/NL/1/00 leader were subsequently confirmed using acombination of polyadenylation and 3′ RACE methods. Furthermore, twoextra nucleotides at the 3′ leader terminus of hMPV NL/1/00 wereidentified.

Vero-grown hMPV NL/1/00 virus was used in this study. As a control, arelated negative sense RNA virus, respiratory syncytial virus (RSV) A2,that has a similar genomic size with identified terminal sequences, wasincluded. Viral RNA was isolated using the QIAamp Viral RNA Mini Kit(Qiagen), following the manufacturer's instructions.

Viral RNA was polyadenylated by incubating the viral RNA with poly (A)polymerase (Ambion) at 37° C. for 1 hour, followed by clean up using aNucAway spin column (Ambion). The viral RNA was then reverse transcribedusing a primer complementary to the poly (A) tail region and the reversetranscriptase, Superscript I (Invitrogen). PCR and Nested PCR reactionswere carried out using hMPV specific primers, juxtaposed to the terminalends, to amplify the desired products with expected sizes for sequencinganalysis. PCR products were further cloned into pCR11 vector using a TAcloning kit (Invitrogen). To reveal the authentic nucleotide sequencesfor the terminus, direct sequencing of PCR DNA as well as the cloned PCRproducts were conducted.

Only hMPV data are shown in FIG. 55. Control experiments, using RSV-A2RNA, indicated that the leader sequences of RSV-A2 remained intact anddetectable with the same approach. Sequencing analyses on PCR productsdirectly (FIG. 55) and on PCR clones both indicated that the leaderregion of hMPV consisted of 5′ ACG CGA AAA AAA CGC GTA TA (SEQ IDNO:485) (expressed as positive sense cDNA orientation) at the 3′ mostproximal 20 nucleotides in the leader sequence. The two newly identifiednucleotides are underlined in FIG. 55.

6.8 Example 8 Serotyping and Subgrouping of MPV Isolates

Virus neutralization assays (see, e.g., Example 16) were used todetermine if the virus isolates of hMPV could be distinguished byserotype or genotype. Virus isolates 00-1 and 99-1 were used toinoculate ferrets in order to raise virus-specific antisera. For the00-1 isolate, ferret and guinea pig specific antisera for the virus weregenerated by experimental intranasal infection of two specific pathogenfree ferrets and two guinea pigs, housed in separate pressurized gloveboxes. Two to three weeks later all the animals were bled by cardiacpuncture, and their sera were used as reference sera. The sera weretested for all previous described viruses with indirect IFA as describedbelow. These antisera, along with antisera prepared using the 99-1isolate, were used in virus neutralization assays with both viruses(Table 10).

TABLE 10 VIRUS NEUTRALIZATION TITERS ISOLATE 00-1 ISOLATE 99-1 PRESERUMFERRET A (00-1) 2 2 FERRET A 22 DPI (00-1) 64 2 PRESERUM FERRET B (99-1)2 2 FERRET B 22 DPI (99-1) 4 64 For isolate 00-1 the titer differs 32(64/2) fold For isolate 99-1 the titer differs 16 (64/4) fold

In addition, six guinea pigs were inoculated with either one of theviruses, i.e., 00-1 and 99-1). RT-PCR assays on nasopharyngeal aspiratesamples showed virus replication from day 2 through day 10 postinfection. At day 70 post infection the guinea pigs were challenged witheither the homologous or the heterologous virus, and in all four casesvirus replication was noticed.

Virus neutralization assays with anti-sera after the first challengeshowed essentially the same results as in the VN assays performed withthe ferrets (>16-fold difference in VN titer).

The results presented in this example confirm the existence of twogenotypes, that correspond to two serotypes of MPV, and show thepossibility of repeated infection with heterologous and homologous virus(Table 11).

TABLE 11 primary virus secondary virus infection replication infectionreplication guinea pig 1-3 00-1 2 out of 3 99-1 1 out of 2 guinea pig4-6 00-1 3 out of 3 00-1 1 out of 3 guinea pig 7-9 99-1 3 out of 3 00-12 out of 2 guinea pig 10-12 99-1 3 out of 3 99-1 1 out of 3 Note: forthe secondary infection guinea pig 2 and 9 were not there any more.

7. DIAGNOSTIC ASSAYS/DETECTION METHODS 7.1 Example 9 DirectImmunofluoresence Assay (DIF) Method

Nasopharyngeal aspirate samples from patients suffering from RTI wereanalyzed by DIF as described (Rothbarth et. al., 1999, J. of Virol.Methods 78:163-169). Samples were stored at −70° C. In short,nasopharyngeal aspirates were diluted with 5 ml Dulbecco MEM(BioWhittaker, Walkersville, Md.) and thoroughly mixed on a vortex mixerfor one minute. The suspension was centrifuged for ten minutes at 840×g.The sediment was spread on a multispot slide (Nutacon, Leimuiden, TheNetherlands) and the supernatant was used for virus isolation. Afterdrying, the cells were fixed in acetone for one minute at roomtemperature. After the slides were washed, they were incubated for 15minutes at 37° C. with commercially available FITC-labeled anti-seraspecific for viruses such as influenza A and B, hRSV and hPIV 1 to 3(Dako, Glostrup, Denmark). After three washings in PBS and one in tapwater, the slides were submerged in a glycerol/PBS solution (Citifluor,UKO, Canterbury, UK) and covered. The slides were then analyzed using aAxioscop fluorescence microscope.

7.2 Example 10 Virus Culture of MPV

The detection of the virus in a cultivated sample from a host is adirect indication of the host's current and/or past exposure orinfection with the virus.

Samples that displayed CPE after the first passage were used toinoculate sub-confluent mono-layers of tMK cells in media in 24 wellplates. Cultures were checked for CPE daily and the media was changedonce a week. Since CPE differed for each isolate, all cultures weretested at day 12 to 14 with indirect WA using ferret antibodies againstthe new virus isolate. Positive cultures were freeze-thawed three times,after which the supernatants were clarified by low-speed centrifugation,aliquoted and stored frozen at −70° C. The 50% tissue culture infectiousdoses (TCID₅₀) of virus in the culture supernatants were determined asdescribed (D. A. Lennette, et al., In: DIAGNOSTIC PROCEDURES FOR VIRAL,RICKETTSIAL, AND CHLAMYDIAL INFECTIONS, 7th ed. (eds. E. H. Lennette, D.A. Lennette, and E. T. Lennette) 3-25; 37-138; 431-463; 481-494; 539-563(American Public Health Association, Washington, 1995)).

7.3 Example 11 Antigen Detection by Indirect Immunofluorescence Assays(IFA)

Antibodies can be used to visualize viral proteins in infected cells ortissues. Indirect immunofluorescence assay (WA) is a sensitive approachin which a second antibody coupled to a fluorescence indicatorrecognizes a general epitope on the virus-specific antibody. IFA is moreadvantageous than DIF because of its higher level of sensitivity.

In order to perform the indirect IFA, collected specimens were dilutedwith 5 ml Dulbecco MEM medium (BioWhittaker, Walkersville, Md.) andthoroughly mixed on a vortex mixer for one minute. The suspension wasthen centrifuged for ten minutes at 840×g. The sediment was spread on amultispot slide. After drying, the cells were fixed in acetone for 1minute at room temperature. Alternatively, virus was cultured on tMKcells in 24 well slides containing glass slides. These glass slides werewashed with PBS and fixed in acetone for 1 minute at room temperature.

Two indirect IFAs were performed. In the first indirect IFA, slidescontaining infected tMK cells were washed with PBS, and then incubatedfor 30 minutes at 37° C. with virus specific antisera. Monoclonalantibodies against influenza A, B and C, hPIV type 1 to 3, and hRSV wereused. For hPIV type 4, mumps virus, measles virus, sendai virus, simianvirus type 5, and New-Castle Disease virus, polyclonal antibodies (RIVM)and ferret and guinea pig reference sera were used. After three washingswith PBS and one wash with tap water, the slides were stained withsecondary antibodies directed against the sera used in the firstincubation. Secondary antibodies for the polyclonal antisera weregoat-anti-ferret (KPL, Guilford, UK, 40 fold diluted), mouse-anti-rabbit(Dako, Glostrup, Denmark, 20 fold diluted), rabbit-anti-chicken (KPL, 20fold dilution) and mouse-anti-guinea pig (Dako, 20 fold diluted).

In the second IFA, after washing with PBS, the slides were incubated for30 minutes at 37° C. with 20 polyclonal antibodies at a dilution of 1:50to 1:100 in PBS. Immunized ferrets and guinea pigs were used to obtainpolyclonal antibodies, but these antibodies can be raised in variousanimals, and the working dilution of the polyclonal antibody can varyfor each immunization. After three washes with PBS and one wash with tapwater, the slides were incubated at 37° C. for 30 minutes with FITClabeled goat-anti-ferret antibodies (KPL, Guilford, UK, 40 folddiluted). After three washes in PBS and one in tap water, the slideswere included in a glycerol/PBS solution (Citifluor, UKO, Canterbury,UK) and covered. The slides were analyzed using an Axioscop fluorescencemicroscope (Carl Zeiss B. V., Weesp, the Netherlands).

7.4 Example 12 Hemagglutination Assays, Chloroform Sensitivity Tests andElectron Microscopy

Different characteristics of a virus can be utilized for the detectionof the virus. For example, many virus contain proteins that can bind toerythrocytes resulting in a lattice. This property is calledhemagglutination and can be used in hemagglutination assays fordetection of the virus. Virus may also be visualized under an electronmicroscope (EM) or detected by PCR techniques.

Hemagglutination assays and chloroform sensitivity tests were performedas described (Osterhaus et al., 1985, Arch. of Virol. 86:239-25;Rothbarth et al., J. of Virol. Methods 78:163-169).

For EM analyses, virus was concentrated from infected cell culturesupernatants in a micro-centrifuge at 4° C. at 17000×g, after which thepellet was resuspended in PBS and inspected by negative contrast EM.

7.5 Example 13 Detection of hMPV/AVP Antibodies of IgG, IgA and IgMClasses

Specific antibodies to viruses rise during the course ofinfection/illness. Thus, detection of virus-specific antibodies in ahost is an indicator of current and/or past infections of the host withthat virus.

The indirect enzyme immunoassay (ETA) was used to detect the IgG classof hMPV antibodies. This assay was performed in microtiter platesessentially as described previously (Rothbarth et al., 1999, J. of Vir.Methods 78:163-169). Briefly, concentrated hMPV was solubilized bytreatment with 1% TRITON® X-100. After determination of the optimalworking dilution by checkerboard titration, it was coated for 16 hoursat room temperature into microtiter plates in PBS. Subsequently, 100 μlvolumes of 1:100 diluted human serum samples in EIA buffer were added tothe wells and incubated for 1 hour at 37° C. Binding of human IgG wasdetected by adding a goat anti-human IgG peroxidase conjugate(Biosource, USA), adding TMB as substrate developed plates and OpticalDensity (OD) was measured at 450 nm. The results were expressed as theS(ignal)/N(egative) ratio of the OD. A serum was considered positive forIgG if the S/N ratio was beyond the negative control plus three timesthe standard.

The hMPV antibodies of the IgM and IgA classes were detected in sera bycapture EIA essentially as described previously (Rothbarth et al., 1999,J. Vir. Methods 78:163-169). For the detection of IgA and IgM,commercially available microtiter plates coated with anti-human IgM orIgA specific monoclonal antibodies were used. Sera were diluted 1:100.After incubation of 1 hour at 37° C., an optimal working dilution ofhMPV was added to each well (100 μl) before incubation for 1 hour at 37°C. After washing, polyclonal anti-hMPV antibody labeled with peroxidasewas added, and the plate was incubated 1 hour at 37° C. Adding TMB as asubstrate the plates were developed, and OD was measured at 450 rim. Theresults were expressed as the S(ignal)/N(egative) ratio of the OD. Apositive result was indicated for IgG when the S/N ratio was beyond thenegative control plus three times the standard.

AVP antibodies were detected in an AVP inhibition assay. The protocolfor the APV inhibition test is included in the APV-Ab SVANOVIR® enzymeimmunoassay that is manufactured by SVANOVA Biotech AB, Uppsala SciencePark Glunten SE-751 83 Uppsala Sweden. The results were expressed as theS(ignal)/N(egative ratio of the OD. A serum was considered positive forIgG, if the S/N ratio was beyond the negative control plus three timesthe standard.

7.6 Example 14 Detection of Antibodies in Humans, Mammals, Ruminants orOther Animals by Indirect IFA

For the detection of virus specific antibodies, infected tMK cells withMPV were fixed with acetone on coverslips (as described above), washedwith PBS and incubated 30 minutes at 37° C. with serum samples at a 1 to16 dilution. After two washes with PBS and one with tap water, theslides were incubated for 30 minutes at 37° C. with FITC-labeledsecondary antibodies to the species used (Dako). Slides were processedas described above.

Antibodies can be labeled directly with a fluorescent dye, which willresult in a direct immunofluorescence assay. FITC can be replaced withany fluorescent dye.

7.7 Example 15 Detection of Antibodies in Humans, Mammals, Ruminants orOther Animals by ELISA

In Paramyxoviridae, the N protein is the most abundant protein, and theimmune response to this protein occurs early in infection. For thesereasons, a recombinant source of the N proteins is preferably used fordeveloping an ELISA assay for detection of antibodies to MPV. Antigenssuitable for antibody detection include any MPV protein that combineswith any MPV-specific antibody of a patient exposed to or infected withMPV virus. Preferred antigens of the invention include those thatpredominantly engender the immune response in patients exposed to MPV,thus, typically are recognized most readily by antibodies of a patient.Particularly preferred antigens include the N, F, M and G proteins ofMPV. Antigens used for immunological techniques can be native antigensor can be modified versions thereof. Well known techniques of molecularbiology can be used to alter the amino acid sequence of a MPV antigen toproduce modified versions of the antigen that may be used in immunologictechniques.

Methods for cloning genes, for manipulating the genes to and fromexpression vectors, and for expressing the protein encoded by the genein a heterologous host are well-known, and these techniques can be usedto provide the expression vectors, host cells, and the for expressingcloned genes encoding antigens in a host to produce recombinant antigensfor use in diagnostic assays. See, e.g., MOLECULAR CLONING, A LABORATORYMANUAL AND CURRENT PROTOCOLS IN MOLECULAR BIOLOGY.

A variety of expression systems may be used to produce MPV antigens. Forinstance, a variety of expression vectors suitable to produce proteinsin E. Coli, B. subtilis, yeast, insect cells, and mammalian cells havebeen described, any of which might be used to produce a MPV antigensuitable to detect anti-MPV antibodies in exposed patients.

The baculovirus expression system has the advantage of providingnecessary processing of proteins, and is therefore preferred. The systemutilizes the polyhedrin promoter to direct expression of MPV antigens.(Matsuura et al., 1987, J. Gen. Virol. 68:1233-1250).

Antigens produced by recombinant baculo-viruses can be used in a varietyof immunological assays to detect anti-MPV antibodies in a patient. Itis well established that recombinant antigens can be used instead ofnatural virus in practically any immunological assay for detection ofvirus specific antibodies. The assays include direct and indirectassays, sandwich assays, solid phase assays such as those using platesor beads among others, and liquid phase assays. Assays suitable includethose that use primary and secondary antibodies, and those that useantibody binding reagents such as protein A. Moreover, a variety ofdetection methods can be used in the invention, including colorimetric,fluorescent, phosphorescent, chemiluminescent, luminescent andradioactive methods.

For example, an indirect IgG EIA using a recombinant N protein (producedwith recombinant baculovirus in insect (Sf9) cells) as antigen can beperformed. For antigen preparation, Sf9 cells are infected with therecombinant baculovirus and harvested 3-7 days post infection. The cellsuspension is washed twice in PBS, pH 7.2, adjusted to a cell density of5.0×10⁶ cells/ml, and freeze-thawed three times. Large cellular debrisis pelleted by low speed centrifugation (500×g for 15 minutes) and thesupernatant is collected and stored at −70° C. until use. Uninfectedcells are processed similarly for negative control antigen.

Once the antigen is prepared, 100 μl of a freeze-thaw lysate is used tocoat microtiter plates at dilutions ranging from 1:50 to 1:1000. Anuninfected cell lysate is run in duplicate wells and serves as anegative control. After incubation overnight, plates are washed twicewith PBS/0.05% TWEEN®. Test sera are diluted 1:50 to 1:200 in ELISAbuffer (PBS, supplemented to 2% with normal goat sera, and with 0.5%bovine serum albumin and 0.1% milk), followed by incubation wells for 1hour at 37° C.

Plates are washed two times with PBS/0.05% TWEEN®. Horseradishperoxidase labeled goat anti-human (or against other species) IgG,diluted 1:3000 to 1:5000 in ELISA buffer, is added to wells, andincubated for 1 hour at 37° C. The plates are then washed two times withPBS/0.05% TWEEN® and once with tap water, incubated for 15 minutes atroom temperature with the enzyme substrate TMB, 3,3′,5,5′tetramethylbenzidine, such as that obtained from Sigma, and the reactionis stopped with 100 μl of 2 M phosphoric acid. Colorimetric readings aremeasured at 450 nm using an automated microtiter plate reader.

7.8 Example 16 Virus Neutralization Assay

When a subject is infected with a virus, an array of antibodies againstthe virus are produced. Some of these antibodies can bind virusparticles and neutralize their infectivity. Virus neutralization assays(VN) are usually conducted by mixing dilutions of serum or monoclonalantibody with virus, incubating them, and assaying for remaininginfectivity with cultured cells, embryonated eggs, or animals.Neutralizing antibodies can be used to define type-specific antigens onthe virus particle, e.g., neutralizing antibodies could be used todefine serotypes of a virus. Additionally, broadly neutralizingantibodies may also exist.

VN assays were performed with serial two-fold dilutions of human andanimal sera starting at an eight-fold dilution. Diluted sera wereincubated for one hour with 100 TCID₅₀ of virus before inoculation oftMK cells grown in 96 well plates, after which the plates werecentrifuged at 840×g. The media was changed after three and six days andIFA was conducted with FTIC-labeled ferret antibodies against MPV 8 daysafter inoculation. The VN titer was defined as the lowest dilution ofthe serum sample resulting in negative IFA and inhibition of CPE in cellcultures.

7.9 Example 17 RNA Isolation

The presence of viruses in a host can also be diagnosed by detecting theviral nucleic acids in samples taken from the host (see, e.g., RT-PCR inExample 18 and RAP-PCR in Example 19).

RNA was isolated from the supernatants of infected cell cultures orsucrose gradient fractions using a High Pure RNA Isolation kit,according to instructions from the manufacturer (Roche Diagnostics,Ahnere, The Netherlands). RNA can also be isolated following otherprocedures known in the art (see, e.g., CURRENT PROTOCOLS IN MOLECULARBIOLOGY, volume 1-3 (1994-1998). Ed. by Ausubel, F. M. et al., Publishedby John Wiley and sons, Inc., USA).

7.10 Example 18 RT-PCR to Detect/Diagnose MPV

Detection of the virus in a biological sample can be done using methodsthat copy or amplify the genomic material of the virus. Virus-specificoligonucleotide sequences for RT-PCR assays on known paramyxoviruses aredescribed below in this Example. A one-step RT-PCR was performed in 50μl reactions containing 50 mM Tris.HCl pH 8.5, 50 mM NaCl, 4 mM MgCl₂, 2mM dithiotreitol, 200 μM each dNTP, 10 units recombinant RNAsin(Promega, Leiden, the Netherlands), 10 units AMV RT (Promega, Leiden,The Netherlands), 5 units Amplitaq Gold DNA polymerase (PE Biosystems,Nieuwerkerk aan de Ijssel, The Netherlands) and 5 μl RNA. Cyclingconditions were 45 minutes at 42° C. and 7 minutes at 95° C. once, 1minute at 95° C., 2 minutes at 42° C. and 3 minutes at 72° C. repeated40 times and 10 minutes at 72° C. once. Primers sequences are providedin the sequence listing. More specifically, the primers used for thenucleoprotein gene were N3 and N4, having nucleotide sequencescorresponding to SEQ ID NOS:28 and 29 respectively, and were used toamplify a 151 nucleotide fragment. The primers used for the matrixprotein gene were M3 and M4, having nucleotide sequences correspondingto SEQ ID NOS:30 and 31 respectively, and were used to amplify a 252nucleotide fragment. The primers used for the polymerase protein genewere L6 and L7, corresponding to SEQ ID NOS:34 and 35 respectively, andwere used to amplify a 173 nucleotide fragment. The primers used for theF protein gene were F7 and F8, corresponding to SEQ ID NOS:32 and 33respectively, and were used to amplify a 221 nucleotide fragment.

Furthermore, probes were used to confirm the presence of hMPV genomesequences. The probe used to detect the M gene had a nucleotide sequencecorresponding to SEQ ID NO:36. The probe used to detect the N gene had anucleotide sequence corresponding to SEQ ID NO:37. The probe used todetect the L gene had a nucleotide sequence corresponding to SEQ IDNO:38.

In another example, primers and probes can be designed based on MPVsequences that are known or obtained through sequencing. Likewise,different sequences of primers and difference buffer and assayconditions to be used for specific purposes would be known to oneskilled in the art.

RT-PCR was used for the detection of known paramyxoviruses as well.Primers for hPIV-1 to 4, mumps, measles, Tupsia, Mapuera, and Hendrawere developed in house and based on alignments of available sequences.Primers for New Castle Disease Virus were taken from Seal, J., J. et al;Clin. Microb., 2624-2630, 1995. Primers for Nipah and generalparamyxovirus-PCR were taken from Chua, et al., 2000, Science, 288. Theprimers used to detect other known paramyxoviruses were as follows:hPIV-1 was detected with primers corresponding to the sequences of SEQID NOS:58 and 59 for the forward and reverse primers respectively,hPIV-2 was detected with primers corresponding to the sequences of SEQID NOS:60 and 61 for the forward and reverse primers respectively,hPIV-3 was detected with primers corresponding to the sequences of SEQID NOS:62 and 63 for the forward and reverse primers respectively,hPIV-4 was detected with primers corresponding to the sequences of SEQID NOS:64 and 65 for the forward and reverse primers respectively, Mumpswas detected with primers corresponding to the sequences of SEQ IDNOS:66 and 67 for the forward and reverser primers respectively, NDV wasdetected with primers corresponding to the sequences of SEQ ID NOS:68and 69 for the forward and reverse primers respectively, Tupaia wasdetected with primers corresponding to the sequences of SEQ ID NOS:70and 71 for the forward and reverse primers respectively, Mapuera wasdetected with primers corresponding to the sequences of SEQ ID NOS:72and 73 for the forward and reverse primers respectively, Hendra wasdetected with primers corresponding to the sequences of SEQ ID NOS:74and 75 for the forward and reverse primers respectively, Nipah wasdetected with primers corresponding to the sequences of SEQ ID NOS:76and 77 for the forward and reverse primers respectively, HRSV wasdetected with primers corresponding to the sequences of SEQ ID NOS:78and 79 for the forward and reverse primers respectively, Measles wasdetected with primers corresponding to the sequences of SEQ ID NOS:80and 81 for the forward and reverse primers respectively, and generalParamyxoviridae viruses were detected with primers corresponding to thesequences of SEQ ID NOS:82 and 83 for the forward and reverse primersrespectively.

7.11 Example 19 RAP-PCR

The genetic material of MPV or another virus can be detected oramplified using primers that hybridize to regions within the genome andthat extend in a particular direction so that the genetic material isamplified. This type of technique is useful when specific sequenceinformation is unavailable or when performing an initial amplificationof genetic material in a sample. One such technique is called RAP-PCR.

RAP-PCR was performed essentially as described (Welsh et al., 1992, NAR20:4965-4970). For the RT reaction, 2 μl of RNA was used in a 10 μlreaction containing 10 ng/μl oligonucleotide, 10 mM dithiotreitol, 500μm each dNTP, 25 mM Tris-HCl pH 8.3, 75 mM KCl and 3 mM MgCl₂. Thereaction mixture was incubated for 5 minutes at 70° C. and 5 minutes at37° C., after which 200 units Superscript RT enzyme (LifeTechnologies)were added. The incubation at 37° C. was continued for 55 minutes andthe reaction was terminated by a 5 minute incubation at 72° C. The RTmixture was diluted to give a 50 μl PCR reaction containing 8 ng/μloligonucleotide, 300 μl each dNTP, 15 mM Tris-HCl pH 8.3, 65 mM KCl, 3.0mM MgCL₂ and 5 units Taq DNA polymerase (FE Biosystems). Cyclingconditions were 5 minutes at 94° C., 5 minutes at 40° C., and 1 minuteat 72° C. once, followed by 1 minute at 94° C., 2 minutes at 56° C. and1 minute at 72° C. repeated 40 times, and 5 minutes at 72° C. once.

Primers used for RAP-PCR were: primer ZF1 with a nucleotide sequencecorresponding to SEQ ID NO:46, primer ZF4 with a nucleotide sequencecorresponding to SEQ ID NO:47, primer ZF7 with a nucleotide sequencecorresponding to SEQ ID NO:48, primer ZF10 with a nucleotide sequencecorresponding to SEQ ID NO:49, primer ZF13 with a nucleotide sequencecorresponding to SEQ ID NO:50, primer ZF16 with a nucleotide sequencecorresponding to SEQ ID NO:51, primer CS1 with a nucleotide sequencecorresponding to SEQ ID NO:52, CS4 with a nucleotide sequencecorresponding to SEQ ID NO:53, primer CS7 with a nucleotide sequencecorresponding to SEQ ID NO:54, primer CS10 with a nucleotide sequencecorresponding to SEQ ID NO:55, primer CS13 with a nucleotide sequencecorresponding to SEQ ID NO:56, and primer CS16 with a nucleotidesequence corresponding to SEQ ID NO:57. Products were run side by sideon a 3% NuSieve agarose gel (FMC BioProducts, Heerhugowaard, TheNetherlands). Differentially displayed fragments specific for MPV werepurified from the gel with a Qiaquick Gel Extraction kit (Qiagen,Leusden, The Netherlands) and cloned in pCR2.1 vector (Invitrogen,Groningen, The Netherlands), according to instructions from themanufacturer. Twenty fragments were successfully purified and sequenced.Sequence homology to APV was found in ten fragments, i.e. fragment 1isolated using the ZF7 primer yielded a 335 by fragment with homology tothe N gene, fragment 2 isolated using the ZF10 primer yielded a 235 byfragment with homology to the N gene, fragment 3 isolated using the ZF10primer yielded a 800 by fragment with homology to the M gene, fragment 4isolated using the CSI primer yielded a 1250 by fragment with homologyto the F gene, fragment 5 isolated using the CS10 primer yielded a 400by fragment with homology to the F gene, fragment 6 isolated using theCS13 primer yielded a 1450 by fragment with homology to the F gene,fragment 7 isolated using primer CS13 yielded a 750 by fragment withhomology to the F gene, fragment 8 isolated using the ZF4 primer yieldeda 780 by fragment with homology to the L gene (protein level), fragment9 isolated using the ZF10 primer yielded a 330 by fragment with homologyto the L gene (protein level), and fragment 10 isolated using the ZF10primer yielded a 250 by fragment with homology to the L gene (proteinlevel).

TaqMan assays can be used to measure the level of expression of a gene.TaqMan assays were adapted to examine the expression of the L-gene andthe N-gene. The primers that were used in these assays are not requiredto be specific to any one of the hMPV groups, however, examples areshown below. Reactions were carried out with a 500 nM concentration of aforward primer, 250 nM concentration of a reverse primer, 250 nMconcentration of an oligonucleotide probe, 25 μl of a universal PCRmastermix (available from ABI), and 5 μl of cDNA in a 50 μl totalreaction volume. Cycling conditions were: a first step of 10 minutes at95° C., followed by a second step of 45 cycles consisting of 30 secondsat 95° C. and 60 seconds at 60° C. on an ABI 7000 sequence detectionsystem.

Other examples of primers for the N gene of hMPV to be used in TaqManassays are as follows: For isolates NL/1/00, BI/1/01, FI/4/01, NL/8/01,and FI/2/01, all of the subgroup A1, primers with the nucleotidesequence of SEQ ID NO:39 could be used. For isolate NL/30/01, of thesubgroup A1, a primer with the nucleotide sequence of SEQ ID NO:40 couldbe used. For isolates NL/22/01 and NL/23/01, of the subgroup A2, aprimer with the nucleotide sequence of SEQ ID NO:41 could be used. Forisolates NL/17/01, of the subgroup A2, a primer with the nucleotidesequence of SEQ ID NO:42 could be used. For isolate NL/17/00, of thesubgroup A2, a primer with the nucleotide sequence of SEQ ID NO:43 couldbe used. For isolates NL/1/99, NL/5/01, NL/21/01, and NL/9/01, of thesubgroup B1, a primer with the nucleotide sequence of SEQ ID NO:44. Forisolates FI/1/01 and FI/10/01, of subgroup B1, a primer with thenucleotide sequence of SEQ ID NO:45 could be used.

A potential probe that can be used for the A1 subgroup corresponds toSEQ ID NO:390, a probe that can be used for the B1 subgroup correspondsto SEQ ID NO:391, and a probe that can be used for the B2 subgroupcorresponds to SEQ ID NO:392.

7.12 Example 20 Sequence Analysis of RAP-PCR Products

After segments are amplified using RAP-PCR, sequence information can beobtained on the amplified segments. In order to do so, it isadvantageous to clone the generated fragments into vectors beforesequencing.

RAP-PCR products cloned in vector pCR2.1 (Invitrogen) were sequencedwith M13-specific oligonucleotides. DNA fragments obtained by RT-PCRwere purified from agarose gels using Qiaquick Gel Extraction kit(Qiagen, Leusden, The Netherlands), and sequenced directly with the sameoligonucleotides used for PCR. Sequence analyses were performed using aDyenamic ET terminator sequencing kit (Amersham Pharmacia Biotech,Roosendaal, The Netherlands) and an ABI 373 automatic DNA sequencer (PEBiosystem). All techniques were performed according to the instructionsof the manufacturer.

7.13 Example 21 Generating Genomic Fragments by RT-PCR

The RAP-PCR method can leave gaps in the sequence that have not beamplified or copied. In order to obtain a complete sequence, thesequence information of the gaps can be obtained using RT-PCR.

To generate PCR fragments spanning gaps A, B and C between the RAP-PCRfragments (FIG. 3), RT-PCR assays were used as described previously onRNA samples isolated from virus isolate 00-1.

The following primers were used to generate fragment A: TR1 designed inthe leader, corresponding to the nucleotide sequence of SEQ ID NO:22 andN1 designed at the 3′ end of the RAP-PCR fragments obtained in N andcorresponding to the sequence of SEQ ID NO:23. The following primerswere used to generate fragment B: N2 designed at the 5′ end of theRAP-PCR fragments obtained in N and corresponding to the nucleotidesequence of SEQ ID NO:24 and M1 designed at the 3′ end of the RAP-PCRfragments obtained in M and corresponding to the nucleotide sequence ofSEQ ID NO:25. The following primers were used to generate fragment C: M2designed at the 5′ end of the RAP-PCR fragment obtained in M andcorresponding to the nucleotide sequence of SEQ ID NO:26 and F1 designedat the 3′ end of the RAP-PCR fragments obtained in F and correspondingto the nucleotide sequence of SEQ ID NO:27.

Fragments were purified after gel electrophoresis and cloned andsequenced as described previously.

7.14 Example 25 Capture Anti-MPV IgM EIA Using a RecombinantNucleoprotein

In order to detect the hMPV virus, an immunological assay that detectsthe presence of the antibodies in a variety of hosts. In one example,antibodies to the N protein are used because it is the most abundantprotein that is produced. This feature is due the transcriptionalgradient that occurs across the genome of the virus.

A capture IgM EIA using the recombinant nucleoprotein or any otherrecombinant protein as antigen can be performed by modification ofassays as previously described by Erdman et al., 1990, J. Clin. Microb.29:1466-1471.

Affinity purified anti-human IgM capture antibody (or against otherspecies), such as that obtained from Dako, is added to wells of amicrotiter plate in a concentration of 250 ng per well in 0.1 Mcarbonate buffer pH 9.6. After overnight incubation at room temperature,the plates are washed two times with PBS/0.05% TWEEN®. 100 μl of testserum diluted 1:200 to 1:1000 in ELISA buffer is added to triplicatewells and incubated for 1 hour at 37° C. The plates are then washed twotimes with in PBS/0.05% TWEEN®.

The freeze-thawed (infected with recombinant virus) Sf21 cell lysate isdiluted 1:100 to 1:500 in ELISA buffer is added to the wells andincubated for 2 hours at 37° C. Uninfected cell lysate serves as anegative control and is run in duplicate wells. The plates are thenwashed three times in PBS/0.05% TWEEN® and incubated for 1 hour at 37°C. with 100 μl of a polyclonal antibody against MPV in an optimaldilution in ELISA buffer. After 2 washes with PBS/0.05% TWEEN®, theplates are incubated with horseradish peroxide labeled secondaryantibody (such as rabbit anti ferret), and the plates are incubated 20minutes at 37° C.

The plates are then washed five times in PBS/0/05% TWEEN®, incubated for15 minutes at room temperature with the enzyme substrate TMB, 3,3,5,5tetramethylbenzidine, as, for instance obtained from “Sigma,” and thereaction is stopped with 100 μl of 2M phosphoric acid. Colormetricreadings are measured at 450 nm using automated microtiter plate reader.

The sensitivities of the capture IgM EIAs using the recombinantnucleoprotein (or other recombinant protein) and whole MPV virus arecompared using acute- and convalescent-phase serum pairs form personswith clinical MPV virus infection. The specificity of the recombinantnucleoprotein capture EIA is determined by testing serum specimens fromhealthy persons and persons with other paramyxovirus infections.

Potential for EIAs for using recombinant MPV fusion and glycoproteinproteins produced by the baculovirus expression.

The glycoproteins G and F are the two transmembranous envelopeglycoproteins of the MPV virion and represent the major neutralizationand protective antigens. The expression of these glycoproteins in avector virus system such as a baculovirus system provides a source ofrecombinant antigens for use in assays for detection of MPV specificantibodies. Moreover, their use in combination with the nucleoprotein,for instance, further enhances the sensitivity of enzyme immunoassays inthe detection of antibodies against MPV.

A variety of other immunological assays (CURRENT PROTOCOLS INIMMUNOLOGY, volume 1-3. Ed. by J. E. Coligan, A. M. Kruisbeek, D. H.Margulies, E. M. Shevach, and W. Strobe, Published by John Wiley andSons, Inc., USA) may be used as alternative methods to those describedhere.

In order to find virus isolates nasopharyngeal aspirates, throat andnasal swabs, broncheo alveolar lavages and throat swabs preferable frombut not limited to humans, carnivores (dogs, cats, seals etc.), horses,ruminants (cattle, sheep, goats etc.), pigs, rabbits, birds (poultry,ostriches, etc.) can be examined. From birds, cloaca and intestinalswabs and droppings can be examined as well. For all samples, serology(antibody and antigen detection etc.), virus isolation and nucleic aciddetection techniques can be performed for the detection of virus.Monoclonal antibodies can be generated by immunizing mice (or otheranimals) with purified MPV or parts thereof (proteins, peptides) andsubsequently using established hybridoma technology (CURRENT PROTOCOLSIN IMMUNOLOGY, Published by John Wiley and sons, Inc., USA).Alternatively, phage display technology can be used for this purpose(Current Protocols in Immunology, Published by John Wiley and sons,Inc., USA). Similarly, polyclonal antibodies can be obtained frominfected humans or animals, or from immunized humans or animals (CURRENTPROTOCOLS IN IMMUNOLOGY, Published by John Wiley and sons, Inc., USA).

The detection of the presence or absence of NS1 and NS2 proteins can beperformed using western-blotting, IFA, immuno precipitation techniquesusing a variety of antibody preparations. The detection of the presenceor absence of NS1 and NS2 genes or homologues thereof in virus isolatescan be performed using PCR with primer sets designed on the basis ofknown NS1 and/or NS2 genes as well as with a variety of nucleic acidhybridization techniques.

To determine whether NS1 and NS2 genes are present at the 3′ end of theviral genome, a PCR can be performed with primers specific for this 3′end of the genome. In our case, we used a primer specific for the 3′untranslated region of the viral genome and a primer in the N ORF. Otherprimers may be designed for the same purpose. The absence of the NS1/NS2genes is revealed by the length and/or nucleotide sequence of the PCRproduct. Primers specific for NS 1 and/or NS2 genes may be used incombination with primers specific for other parts of the 3′ end of theviral genome (such as the untranslated region or N, M or F ORFs) toallow a positive identification of the presence of NS1 or NS2 genes. Inaddition to PCR, a variety of techniques such as molecular cloning,nucleic acid hybridization may be used for the same purpose.

8. CELL CULTURE SYSTEMS AND ANIMAL MODELS FOR MPV AND RECOMBINANTENGINEERING OF MPV 8.1 Example 22 HMPV Growth in Different Cell Lines

Virus isolates can be cultured in different cell lines in order toexamine characteristics of each virus. For example, the infectivity ofdifferent virus isolates can be characterized and distinguished on thebasis of titer levels measured in culture. Alternatively, cells can beused to propagate or amplify strains of the virus in culture for furtheranalysis.

In one example, tertiary monkey kidney cells were used to amplify hMPV.However, tertiary monkey kidney cells are derived from primary cellswhich may only be passaged a limited number of times and have beenpassaged three times in vivo. It was not known which kind ofimmortalized cell line would support hMPV virus growth to high titers. Anumber of monkey cell lines such as Vero, LLC-MK2, HEp-2, and lungfibroblast (LF1043) cells, were tested to see whether they could supporthMPV virus replication (Table 12). Trypsin used was TPCK-trypsin(Worthington) at a concentration of 0.001 mg/ml. The growth of thisvirus in fertilized 10 day old chicken eggs was also tested. Theinfected eggs were incubated for 2 and 3 days at 37° C. prior to AFharvest. Virus titers were determined by plaque assay of infected celllysates on Vero cells without trypsin, incubated for 10 days at 35° C.,and immunostained using the guinea pig hMPV antiserum. The resultsshowed that Vero cells and LLC-MK2 cells were the cell substrates mostsuitable for hMPV virus replication, resulting in virus stock titers of10⁶-10⁷ pfu/ml. These titers were similar to those obtained from tMKcells. The addition of trypsin at a concentration of 0.01 mg/ml did notincrease virus titers appreciably (Table 12).

TABLE 12 HMPV VIRUS GROWTH IN DIFFERENT CELL LINES Trypsin used to Virustiters on Cell Substrate grow virus Vero cells (pfu/ml) Vero yes 2.1 ×10⁷ no 1.1 × 10⁷ LLC-MK2 yes 2.3 × 10⁵ Hep-2 yes cells died LF 1043(HEL) yes no virus recovered no no virus recovered tMK yes 1.0 × 10⁷eggs (10 days) no no virus recovered

In order to study the virus kinetics of hMPV viral growth in Vero cells,a growth curve was performed using an MOI of 0.1 (FIG. 23). Cells andcell supernatants were harvested every 24 hours, and analyzed by plaqueassay for quantification of virus titers. The results showed that at day5, near peak titers of hMPV were observed. The absolute peak titer of5.4 log₁₀ pfu/ml was achieved on Day 8. The virus titer was very stableup to day 10. A growth curve carried out at the same time with solelythe cell supernatants, showed only very low virus titers. This datademonstrated that hMPV replication, under the conditions used (MOI of0.1) peaked on day 8 post-infection and that hMPV was largely, acell-associated RNA virus.

TRANSFECTION OF 293 CELLS: 293 cells (human kidney epithelial cells)were passed in DMEM and supplemented with FCS (10%), L-Glutamine (1:100)and Pen/Strep (1:100) and split 1:10 every 3-4 days. Care was taken notto let the cells grow to confluency in order to enhancetransfectability. Cells were not very adherent; a very brief (2 minutesor less) incubation in Trypsin-EDTA was usually sufficient to releasethem from plastic surfaces. Cells were diluted in culture mediaimmediately after trypsin-treatment.

Cells were split the day before transfection. Cell confluencyapproximated 50-75% when transfected. Gelatinized plasticware was usedto prevent cells from detaching throughout the transfection procedure.Plates or flasks were covered with 0.1% gelatinin (1:20 dilution of 2%stock) for 10 minutes and rinsed one time with PBS once. To achieve thecorrect cell density, cells were used at a concentration of 1×10⁷ cellsper T75 flask or 100 mm plate (in 10 ml) or 1×10⁶ cells per well of a6-well plate (in 2 ml).

Transfection lasted for a minimum of 7 hours, however, it was preferableto allow the transfection to occur overnight. The following werecombined in a sterile tube: 30 mg DNA with 62 ml 2 M CaCl₂ and H₂O to500 ml (T75) or 3 mg DNA with 6.2 ml 2 M CaCl₂ and H₂O to 50 ml (6-wellplate); with brief mixing. Addition of 500 or 50 ml 2×HBS occurreddropwise and the solutions were allowed to mix for 5 minutes until aprecipitate formed. Gentle care was used, i.e. no vortexing was applied.The old media was replaced with fresh prewarmed media (10 ml per T75flask or 1 ml per well of a 6-well plate. The DNA was mixed carefully byblowing air bubbles through the tube with a Gilson pipet and theprecipitate was added dropwise to the media covering the cells. Thecells were incubated in a 37° C. CO₂ atmosphere.

The cells appeared to be covered with little specks (the precipitate).The transfection media was removed from the cells, and the cells wererinsed carefully with PBS, and then replaced with fresh media.

The cells were incubated in a 37° C. CO₂ atmosphere until needed,usually between 8-24 hours.

A 10× stock of HBS was prepared with 8.18% NaCl, 5.94% Hepes and 0.2%Na₂HPO₄ (all w/v). The solution was filter sterilized and stored at 4°C. A 2× solution was prepared by diluting the 10× stock with H₂O andadjusting the pH to 7.12 with 1 M NaOH. The solution was stored inaliquots at −20° C. Care was taken to exactly titrate the pH of thesolution. The pH was adjusted immediately before the solution was usedfor the transfection procedure.

8.2 Example 23 Minireplicon Construct of MPV

Minireplicon constructs can be generated to contain an antisensereporter gene. An example of a minireplicon, CAT-hMPV, is shown in FIGS.24A and 24B. The leader and trailer sequences that were used for thegeneration of the minireplicon construct are shown in FIG. 26. Forcomparison, an alignment of APV, RSV and PIV3 leader and trailersequences are also shown in FIG. 26.

Two versions of the minireplicon constructs were tested: one withterminal AC residues at the leader end (Le+FAC), and one withoutterminal AC residues at the leader end (Le-AC). The two constructs wereboth functional in the assay (FIG. 25). It can be seen in FIG. 25 thatmuch higher CAT expression occurred after 48 hours than after 24 hours.After 48 hours, around 14 ng CAT per 500,000 cells transfected wasobserved. This experiment was entirely plasmid driven: the minirepliconwas cotransfected with a T7 polymerase plasmid, and the N, P, L, M2.1genes were expressed from pCITE-2a/3a (the pCite plasmids have a T7promoter followed by the IBES element derived from theencephalomyocarditis virus (EMCV)). The CAT expression was completelyabolished when L, P and N were excluded. A significant reduction in CATexpression was noted when M2.1 expression was excluded from the vector.

The specificity (attributes to heterologous viruses) and the effect ofthe terminal residues of the leader (attributes to homologous virus) ofthe minireplicon system can also be tested by superinfecting theminireplicon-transfected cells with hMPV polymerase components (NL/1/00and NL/1/99) or polymerase components from APV-A, APV-C, RSV or PIV. Thedifferent amount of each of the six plasmids can also be tested in orderto determine the optimal conditions.

Other reporter genes can be used instead of CAT. In other examples, GFPcan be inserted into the minireplicon construct instead of CAT.

8.3 Example 24 Generation of Full Length Infectious cDNA

Full length cDNAs that express the genes of the hMPV virus can beconstructed so that infectious viruses can be produced. For example, acDNA encoding all of the genes or all of the essential genes of hMPV canbe constructed; the genome can then be expressed to produce infectiousviruses.

In order to genetically manipulate hMPV, the genome of this RNA viruswas cloned. For the 00-1 isolate of hMPV, eight PCR fragments varying inlength from 1-3 kb were generated (FIG. 27). The PCR fragments weresequenced and analyzed for sequence errors by comparison to the hMPVsequence deposited in Genbank. Two silent mutations (nucleotide 5780ile:ile in the SH gene, nucleotide 12219 cys:cys in the L gene) were notcorrected. Another change in the L gene at nucleotide 8352 (trp:leu) wasnot changed since this mutation was observed in two independentlygenerated PCR fragments (C and H), as well as in the hMPV 99-1 sequence.Similarly, a 5 nucleotide insertion at nucleotide 4715 in the F-M2intergenic region was not corrected. Both of these changes may bereflected in the wild type sequence of hMPV. In contrast, at nucleotide1242, a single A residue was removed in the N-P intergenic region; atnucleotide 3367, a ser:pro was corrected in the F gene; at nucleotide6296, an asp:val was changed in the G gene; and at nucleotide 7332 astop codon was changed to a glu in the L gene.

Restriction maps of different isolates of hMPV are shown in FIG. 28. Therestriction sites can be used to assemble the full-length construct.

The eight corrected PCR fragments were then assembled in sequence,taking advantage of unique restriction enzyme sites (FIG. 29). A geneticmarker was introduced at nucleotide 75 generating an AflII restrictionenzyme site without altering the amino acid sequence. A uniquerestriction enzyme site, XhoI, was added at the 3′ end of the hMPVsequence. A phage T7 polymerase promoter followed by two G residues wasalso added to the 3′ end of the hMPV sequence. At the 5′ end of the hMPVgenome, a Hepatitis delta ribozyme sequence and BssHII restrictionenzyme site were added.

Helper plasmids encoding the hMPV L, N, P and M2-1 gene in a pCITEplasmid were also generated. Once the full-length hMPV cDNA wasgenerated, virus recovery by reverse genetics was performed in Verocells using fowl-pox T7 or MVA-T7 as a source of T7 polymerase.

8.4 Example 26 Infection of Animal Hosts with Subtypes of hMPV

Animal hosts can be infected in order to characterize the virulence ofMPV strains. For example, different hosts can be used in order todetermine how infectious each strain is in an organism.

A small animal model for hMPV had not been identified. Balb/c mice,cotton rats, and Syrian Golden hamsters were infected with hMPV using adose of 1.3×10⁶ pfu/animal. The animals were inoculated intranasallywith 1.3×10⁶ pfu of hMPV in a 0.1 ml volume. The tissue samples werequantified by plaque assays that were immunostained on Day 9 with thehMPV guinea pig antiserum. Four days post-infection, the animals weresacrificed, and the nasal turbinates and lungs were isolated andquantified for hMPV titers by plaque assays that were immunostained(Table 13).

TABLE 13 HMPV TITERS IN INFECTED ANIMALS Mean virus titer on day 4post-infection (log₁₀ PFU/g Number of tissue +/− Standard Error Animalsanimals Nasal turbinates Lungs mice (Balb c) 6 2.7 +/− 0.4 2.2 +/− 0.6cotton rats 5 <1.7 +/− 0.0  <1.8 +/− 0.0  Syrian Golden hamsters 6 5.3+/− 0.2 2.3 +/− 0.6

The results showed that hMPV replicated to high titers in Syrian Goldenhamsters. Titers of 5.3 and 2.3 log₁₀ pfu/g tissue were obtained in thenasal turbinates and lungs, respectively. hMPV did not replicate to anyappreciable titer levels in the respiratory tracts of cotton rats. Miceshowed titers of 2.7 and 2.2 log₁₀ pfu/g tissue in the upper and lowerrespiratory tracts, respectively. These results suggested that SyrianGolden hamsters would be a suitable small animal model to study hMPVreplication and immunogenicity as well as to evaluate hMPV vaccinecandidates.

INFECTION OF GUINEA PIGS. Two virus isolates, 00-1 (subtype A) and 99-1(subtype B), were used to inoculate six guinea pigs per subtype(intratracheal, nose and eyes). Six guinea pigs were infected with hMPV00-1 (10e-6.5 TCID₅₀). Six guinea pigs were infected with hMPV 99-1(10e-4.1 TCID₅₀). The primary infection was allowed to progress forfifty-four days when the guinea pigs were inoculated with the homologousand heterologous subtypes (10e4 TCID₅₀/ml), i.e., two guinea pigs had aprimary infection with 00-1 and a secondary infection with 99-1 in orderto achieve a heterologous infection, three guinea pigs had a primaryinfection with 00-1 and a secondary infection with 00-1 to achieve ahomologous infection, two guinea pigs had a primary infection with 99-1and a secondary infection with 00-1 to achieve a heterologous infectionand three guinea pigs had a primary infection with 99-1 and a secondaryinfection with 99-1 to achieve a homologous infection.

Throat and nose swabs were collected for 12 days (primary infection) or8 days (secondary infection) post infection, and were tested for thepresence of the virus by RT-PCR assays. The results (FIG. 32) of theRT-PCR assays showed that guinea pigs inoculated with virus isolate 00-1showed infection of the upper respiratory tract on days 1 through 10post infection. Guinea pigs inoculated with 99-1 showed infection of theupper respiratory tract day 1 to 5 post infection. Infection of guineapigs with 99-1 appeared to be less severe than infection with 00-1. Asecond inoculation of the guinea pigs with the heterologous virus, ascommented on above, resulted in re-infection in 3 out of 4 of the guineapigs. Likewise, reinfection in the case of the homologous virus occurredin 2 out of 6 guinea pigs. Little or no clinical symptoms were noted inthose animals that became re-infected, and no clinical symptoms wereseen in those animals that were protected against the re-infections,demonstrating that even with the wild-type virus, a protective effectdue to the first infection may have occurred. This also showed thatheterologous and homologous isolates could be used as a vaccine.

Both subtypes of hMPV were able to infect guinea pigs, althoughinfection with subtype B (99-1) seemed less severe, i.e., the presenceof the virus in nose and throat was for a shorter period than infectionwith subtype A (00-1). This may have been due to the higher dose givenfor subtype A, or to the lower virulence of subtype B. Although thepresence of pre-existing immunity did not completely protect againstre-infection with both the homologous and heterologous virus, theinfection appeared to be less prominent, in that a shorter period ofpresence of virus was noted and not all animals became virus positive.

The serology of guinea pigs that were infected with both subtypes ofhMPV was examined. At days 0, 52, 70, 80, 90, 110, 126 and 160, serawere collected from the guinea pigs and tested at a 1:100 dilution in awhole virus ELISA against 00-1 and 99-1 antigens. (See FIGS. 33A and Bshowing the IgG response against 00-1 and 99-1 for each individualguinea pig. See also FIG. 34 showing the specificity of the 00-1 and99-1 ELISA but note that only data from homologous reinfected guineapigs was used. See also FIG. 35 showing the mean IgG response against00-1 and 99-1 ELISA of three homologous, i.e. 00-1 and 00-1, twohomologous, i.e., 99-1 and 99-1, two heterologous, i.e., 99-1 and 00-1,and 2 heterologous, i.e., 00-1 and 99-1 infected guinea pigs.)

Only a minor difference in response to the two different ELISAs wasobserved. Whole virus ELISA against 00-1 or 99-1 could not be used todiscriminate between the two subtypes.

The reactivity of sera raised against hMPV in guinea pigs with APVantigen was examined. Sera were collected from the infected guinea pigsand tested with an APV inhibition ELISA. (See FIG. 36, showing the meanpercentage of APV inhibition of hMPV infected guinea pigs.) Sera raisedagainst hMPV in guinea pigs reacted in the APV inhibition test in amanner similar to their reaction in the hMPV IgG ELISAs. Sera raisedagainst 99-1 revealed a lower percentage of inhibition in the APVinhibition ELISA than sera raised against 00-1. Guinea pigs infectedwith 99-1 may have had a lower titer than that seen in the hMVP ELISAs.Alternatively, the cross-reaction of 99-1 with APV could have been lessthan that of 00-1. Nevertheless, the APVAb inhibition ELISA could beused to detect hMPV antibodies in guinea pigs.

Virus neutralization assays were performed with sera raised against hMPVin guinea pigs. Sera were collected at day 0, day 52, day 70 and day 80post infection and used in a virus cross-neutralization assay with 00-1,99-1, and APV-C. The starting dilution used was 1 to 10 and 100 TCID50virus per well. After neutralization, the virus was exposed to tMK cells(15 mm.) and centrifuged at 3500 RPM, after which the media wasrefreshed. The APV cultures were grown for 4 days and the hMPV cultureswere grown for 7 days. Cells were fixed with 80% acetone, and IFAs wereconducted with labeled monkey-anti hMPV. Wells that were negative uponstaining were defined as the neutralizing titer. For each virus, a10-log titration of the virus stock and a 2 fold titration of theworking solution was included. (See FIG. 37 showing the virusneutralization titers of 00-01 and 99-1 infected guinea pigs against00-1, 99-1, and APV-C.)

INFECTION OF CYNOMOLOGOUS MACAGUES. Virus isolates 00-1 (subtype A) and99-1 (subtype B) (1e5 TCID₅₀) was used to inoculate two cynomologousmacaques per subtype (intratracheal, nose and eyes). Six months afterthe primary infection, the macaques were inoculated for the second timewith 00-1. Throat swabs were collected for 14 days (primary infection)or 8 days (secondary infection) post infection, and were tested forpresence of the virus by RT-PCR assays (FIG. 38).

Cynomologous macaques inoculated with virus isolate 00-1 showedinfection of the upper respiratory tract day 1 to 10 post infection.Clinical symptoms included a suppurative rhinitis. A second inoculationof the macaques with the homologous virus results in re-infection, asdemonstrated by PCR, however, no clinical symptoms were seen.

Sera were collected from the macaques that received 00-1 during sixmonths after the primary infection (re-infection occurred at day 240 formonkey 3 and day 239 for monkey 6). Sera were used to test for thepresence of IgG (FIG. 39B) antibodies against either 00-1 or APV, andfor the presence of IgA and IgM antibodies against 00-1 (FIG. 39A).

Two macaques were successfully infected with 00-1 and in the presence ofantibodies against 00-1 were reinfected with the homologous virus. Theresponse to IgA and IgM antibodies showed the raise in IgM antibodiesafter the first infection, and the absence of it after the reinfection.IgA antibodies were only detected after the re-infection, showing theimmediacy of the immune response after a first infection. Sera raisedagainst hMPV in macaques that were tested in an APV inhibition ELISAshowed a similar response as to the hMPV IgG ELISA.

Antibodies to hMPV in cynomologous macaques were detected with the APVinhibition ELISA using a similar sensitivity as that with the hMPVELISA, and therefore the APV inhibition EIA was suitable for testinghuman samples for the presence of hMPV antibodies.

Virus cross-neutralization assays were preformed on sera collected fromhMPV infected cynomologous macaques. The sera were taken from day 0 today 229 post primary infection and showed only low virus neutralizationtiters against 00-1 (0-80), the sera taken after the secondary infectionshowed high neutralization titers against 00-1, i.e., greater than 1280.Only sera taken after the secondary infection showed neutralizationtiters against 99-1 (80-640), and none of the sera were able toneutralize the APV C virus. There was no cross reaction between APV-Cand hMPV in virus cross-neutralization assays, however, there was across reaction between 00-1 and 99-1 after a boost of the antibodyresponse.

INFECTION OF HUMANS. The sera of patients ranging in ages under sixmonths or greater than twenty years of age were previously tested usingIFA and virus neutralization assays against 00-1. These sera were testedfor the presence of IgG, IgM and IgA antibodies in an ELISA against00-1. The samples were also tested for their ability to in inhibit theAPV ELISA. A comparison of the use of the hMPV ELISA and the APVinhibition ELISA for the detection of IgG antibodies in human sera wasmade and a strong correlation between the IgG hMPV test and the APV-Abtest was noted, therefore the APV-Ab test was essentially able to detectIgG antibodies to hMPV in humans (FIG. 40).

INFECTION OF POULTRY. The APV inhibition ELISA and the 00-1 ELISA wereused to test chickens for the presence of IgG antibodies against APV.Both the hMPV ELISA and the APV inhibition ELISA detected antibodiesagainst APV.

8.5 Example 27 APV as a Vaccine in Humans

APV can be used as a vaccine in humans to prevent infection by a humanMPV, or to reduce the infectivity of human MPV in human hosts. Thevaccine can be a whole APV or a chimeric or recombinant version orderivative thereof, that is comprised of heterologous sequences ofanother metapneumovirus in addition to sequences of APV. The genome ofAPV can be used as a backbone to create a recombinant virus vaccine. Forexample, a vaccine can be made where the F-gene and/or the G-gene of APVis substituted by the F-gene or the G-gene of human MPV. Alternatively,a vaccine can be made that includes sequences from PIV substituted foror added to sequences of an APV backbone. For more on the constructionof a recombinant/chimeric vaccine, see, e.g., Construction of theRecombinant cDNA and RNA.

The vaccine can be administered to a candidate by a variety of methodsknown to those skilled in the art, (see, Section 5.13, infra) includingbut not limited to, subcutaneous injection, intranasal administration,or inhalation. The viruses and/or vaccines of the invention areadministered at a starting dosage of at least between 10³ TCID₅₀ and 10⁶TCID₅₀. The viruses and/or vaccines are administered in either single ormultiple dosages, e.g., a primary dose can be administered with one ormore subsequent or booster doses administered at periodic time intervalsthroughout the host life. In a clinical trial, the replication rate ofthe virus can be used as an index to adjust the dosage of the vaccine sothat an effective dosage regimen can be determined. A comparison can bemade between the replication rate of the virus in the study populationand a predetermined rate that is known to be effective.

The present invention is not to be limited in scope by the specificdescribed embodiments that are intended as single illustrations ofindividual aspects of the invention, and any constructs, viruses orenzymes that are functionally equivalent are within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description and accompanying drawings.Such modifications are intended to fall within the scope of the appendedclaims.

8.6 Example 28 MPV as a Vaccine in Birds

Human MPV can be used as a vaccine in birds to prevent infection by anAPV, or to reduce the infectivity of APV in avian hosts. The vaccine canbe a whole MPV or a chimeric or recombinant version or derivativethereof, that is comprised of heterologous sequences of anothermetapneumovirus in addition to sequences of MPV. The genome of human MPVcan be used as a backbone to create a recombinant virus vaccine. Forexample, a vaccine can be made where the F-gene and/or the G-gene ofhuman MPV is substituted by the F-gene or the G-gene of APV. For more onthe construction of a recombinant/chimeric vaccine, see, e.g.,Construction of the Recombinant cDNA and RNA.

The vaccine can be administered to a candidate by a variety of methods,including but not limited to, subcutaneous injection, intranasaladministration, or inhalation. The viruses and/or vaccines of theinvention are administered at a starting dosage of at least between 10³TCID₅₀ and 10⁶ TCID₅₀. The viruses and/or vaccines are administered ineither single or multiple dosages, e.g., a primary dose can beadministered with one or more subsequent or booster doses administeredat periodic time intervals throughout the host life. In a clinicaltrial, the replication rate of the virus can be used as an index toadjust the dosage of the vaccine so that an effective dosage regimen canbe determined. A comparison can be made between the replication rate ofthe virus in the study population and a predetermined rate that is knownto be effective.

Various publications are cited herein, the disclosures of which areincorporated by reference in their entireties.

TABLE 14 LEGEND FOR SEQUENCE LISTING SEQ ID NO: 1 Human metapneumovirusisolate 00-1 matrix protein (M) and fusion protein (F) genes SEQ ID NO:2 Avian pneumovirus fusion protein gene, partial cds SEQ ID NO: 3 Avianpneumovirus isolate 1b fusion protein mRNA, complete cds SEQ ID NO: 4Turkey rhinotracheitis virus gene for fusion protein (F1 and F2subunits), complete cds SEQ ID NO: 5 Avian pneumovirus matrix protein(M) gene, partial cds and Avian pneumovirus fusion glycoprotein (F)gene, complete cds SEQ ID NO: 6 paramyxovirus F protein hRSV B SEQ IDNO: 7 paramyxovirus F protein hRSV A2 SEQ ID NO: 8 human metapneumovirus01-71 (partial sequence) SEQ ID NO: 9 Human metapneumovirus isolate 00-1matrix protein (M) and fusion protein (F) genes SEQ ID NO: 10 Avianpneumovirus fusion protein gene, partial cds SEQ ID NO: 11 Avianpneumovirus isolate 1b fusion protein mRNA, complete cds SEQ ID NO: 12Turkey rhinotracheitis virus gene for fusion protein (F1 and F2subunits), complete cds SEQ ID NO: 13 Avian pneumovirus fusionglycoprotein (F) gene, complete cds SEQ ID NO: 14 Turkey rhinotracheitisvirus (strain CVL14/1) attachment protein (G) mRNA, complete cds SEQ IDNO: 15 Turkey rhinotracheitis virus (strain 6574) attachment protein(G), complete cds SEQ ID NO: 16 Turkey rhinotracheitis virus (strainCVL14/1) attachment protein (G) mRNA, complete cds SEQ ID NO: 17 Turkeyrhinotracheitis virus (strain 6574) attachment protein (G), complete cdsSEQ ID NO: 18 isolate NL/1/99 (99-1) HMPV (Human Metapneumovirus) cDNAsequence SEQ ID NO: 19 isolate NL/1/00 (00-1) HMPV cDNA sequence SEQ IDNO: 20 isolate NL/17/00 HMPV cDNA sequence SEQ ID NO: 21 isolate NL/1/94HMPV cDNA sequence SEQ ID NO: 22 RT-PCR primer TR1 SEQ ID NO: 23 RT-PCRprimer N1 SEQ ID NO: 24 RT-PCR primer N2 SEQ ID NO: 25 RT-PCR primer M1SEQ ID NO: 26 RT-PCR primer M2 SEQ ID NO: 27 RT-PCR primer F1 SEQ ID NO:28 RT-PCR primer N3 SEQ ID NO: 29 RT-PCR primer N4 SEQ ID NO: 30 RT-PCRprimer M3 SEQ ID NO: 31 RT-PCR primer M4 SEQ ID NO: 32 RT-PCR primer F7SEQ ID NO: 33 RT-PCR primer F8 SEQ ID NO: 34 RT-PCR primer L6 SEQ ID NO:35 RT-PCR primer L7 SEQ ID NO: 36 Oligonucleotide probe M SEQ ID NO: 37Oligonucleotide probe N SEQ ID NO: 38 Oligonucleotide probe L SEQ ID NO:39 TaqMan primer and probe sequences for isolates NL/1/00, BI/1/01,FI/4/01, NL/8/01, FI/2/01 SEQ ID NO: 40 TaqMan primer and probesequences for isolates NL/30/01 SEQ ID NO: 41 TaqMan primer and probesequences for isolates NL/22/01 and NL/23/01 SEQ ID NO: 42 TaqMan primerand probe sequences for isolate NL/17/01 SEQ ID NO: 43 TaqMan primer andprobe sequences for isolate NL/17/00 SEQ ID NO: 44 TaqMan primer andprobe sequences for isolates NL/9/01, NL/21/01, and NL/5/01 SEQ ID NO:45 TaqMan primer and probe sequences for isolates FI/1/01 and FI/10/01SEQ ID NO: 46 Primer ZF1 SEQ ID NO: 47 Primer ZF4 SEQ ID NO: 48 PrimerZF7 SEQ ID NO: 49 Primer ZF10 SEQ ID NO: 50 Primer ZF13 SEQ ID NO: 51Primer ZF16 SEQ ID NO: 52 Primer CS1 SEQ ID NO: 53 Primer CS4 SEQ ID NO:54 Primer CS7 SEQ ID NO: 55 Primer CS10 SEQ ID NO: 56 Primer CS13 SEQ IDNO: 57 Primer CS16 SEQ ID NO: 58 Forward primer for amplification ofHPIV-1 SEQ ID NO: 59 Reverse primer for amplification of HPIV-1 SEQ IDNO: 60 Forward primer for amplification of HPIV-2 SEQ ID NO: 61 Reverseprimer for amplification of HPIV-2 SEQ ID NO: 62 Forward primer foramplification of HPIV-3 SEQ ID NO: 63 Reverse primer for amplificationof HPIV-3 SEQ ID NO: 64 Forward primer for amplification of HPIV-4 SEQID NO: 65 Reverse primer for amplification of HPIV-4 SEQ ID NO: 66Forward primer for amplification of Mumps SEQ ID NO: 67 Reverse primerfor amplification of Mumps SEQ ID NO: 68 Forward primer foramplification of NDV SEQ ID NO: 69 Reverse primer for amplification ofNDV SEQ ID NO: 70 Forward primer for amplification of Tupaia SEQ ID NO:71 Reverse primer for amplification of Tupaia SEQ ID NO: 72 Forwardprimer for amplification of Mapuera SEQ ID NO: 73 Reverse primer foramplification of Mapuera SEQ ID NO: 74 Forward primer for amplificationof Hendra SEQ ID NO: 75 Reverse primer for amplification of Hendra SEQID NO: 76 Forward primer for amplification of Nipah SEQ ID NO: 77Reverse primer for amplification of Nipah SEQ ID NO: 78 Forward primerfor amplification of HRSV SEQ ID NO: 79 Reverse primer for amplificationof HRSV SEQ ID NO: 80 Forward primer for amplification of Measles SEQ IDNO: 81 Reverse primer for amplification of Measles SEQ ID NO: 82 Forwardprimer to amplify general paramyxoviridae viruses SEQ ID NO: 83 Reverseprimer to amplify general paramyxoviridae viruses SEQ ID NO: 84 G-genecoding sequence for isolate NL/1/00 (A1) SEQ ID NO: 85 G-gene codingsequence for isolate BR/2/01 (A1) SEQ ID NO: 86 G-gene coding sequencefor isolate FL/4/01 (A1) SEQ ID NO: 87 G-gene coding sequence forisolate FL/3/01 (A1) SEQ ID NO: 88 G-gene coding sequence for isolateFL/8/01 (A1) SEQ ID NO: 89 G-gene coding sequence for isolate FL/10/01(A1) SEQ ID NO: 90 G-gene coding sequence for isolate NL/10/01 (A1) SEQID NO: 91 G-gene coding sequence for isolate NL/2/02 (A1) SEQ ID NO: 92G-gene coding sequence for isolate NL/17/00 (A2) SEQ ID NO: 93 G-genecoding sequence for isolate NL/1/81 (A2) SEQ ID NO: 94 G-gene codingsequence for isolate NL/1/93 (A2) SEQ ID NO: 95 G-gene coding sequencefor isolate NL/2/93 (A2) SEQ ID NO: 96 G-gene coding sequence forisolate NL/3/93 (A2) SEQ ID NO: 97 G-gene coding sequence for isolateNL/1/95 (A2) SEQ ID NO: 98 G-gene coding sequence for isolate NL/2/96(A2) SEQ ID NO: 99 G-gene coding sequence for isolate NL/3/96 (A2) SEQID NO: 100 G-gene coding sequence for isolate NL/22/01 (A2) SEQ ID NO:101 G-gene coding sequence for isolate NL/24/01 (A2) SEQ ID NO: 102G-gene coding sequence for isolate NL/23/01 (A2) SEQ ID NO: 103 G-genecoding sequence for isolate NL/29/01 (A2) SEQ ID NO: 104 G-gene codingsequence for isolate NL/3/02 (A2) SEQ ID NO: 105 G-gene coding sequencefor isolate NL/1/99 (B1) SEQ ID NO: 106 G-gene coding sequence forisolate NL/11/00 (B1) SEQ ID NO: 107 G-gene coding sequence for isolateNL/12/00 (B1) SEQ ID NO: 108 G-gene coding sequence for isolate NL/5/01(B1) SEQ ID NO: 109 G-gene coding sequence for isolate NL/9/01 (B1) SEQID NO: 110 G-gene coding sequence for isolate NL/21/01 (B1) SEQ ID NO:111 G-gene coding sequence for isolate NL/1/94 (B2) SEQ ID NO: 112G-gene coding sequence for isolate NL/1/82 (B2) SEQ ID NO: 113 G-genecoding sequence for isolate NL/1/96 (B2) SEQ ID NO: 114 G-gene codingsequence for isolate NL/6/97 (B2) SEQ ID NO: 115 G-gene coding sequencefor isolate NL/9/00 (B2) SEQ ID NO: 116 G-gene coding sequence forisolate NL/3/01 (B2) SEQ ID NO: 117 G-gene coding sequence for isolateNL/4/01 (B2) SEQ ID NO: 118 G-gene coding sequence for isolate UK/5/01(B2) SEQ ID NO: 119 G-protein sequence for isolate NL/1/00 (A1) SEQ IDNO: 120 G-protein sequence for isolate BR/2/01 (A1) SEQ ID NO: 121G-protein sequence for isolate FL/4/01 (A1) SEQ ID NO: 122 G-proteinsequence for isolate FL/3/01 (A1) SEQ ID NO: 123 G-protein sequence forisolate FL/8/01 (A1) SEQ ID NO: 124 G-protein sequence for isolateFL/10/01 (A1) SEQ ID NO: 125 G-protein sequence for isolate NL/10/01(A1) SEQ ID NO: 126 G-protein sequence for isolate NL/2/02 (A1) SEQ IDNO: 127 G-protein sequence for isolate NL/17/00 (A2) SEQ ID NO: 128G-protein sequence for isolate NL/1/81 (A2) SEQ ID NO: 129 G-proteinsequence for isolate NL/1/93 (A2) SEQ ID NO: 130 G-protein sequence forisolate NL/2/93 (A2) SEQ ID NO: 131 G-protein sequence for isolateNL/3/93 (A2) SEQ ID NO: 132 G-protein sequence for isolate NL/1/95 (A2)SEQ ID NO: 133 G-protein sequence for isolate NL/2/96 (A2) SEQ ID NO:134 G-protein sequence for isolate NL/3/96 (A2) SEQ ID NO: 135 G-proteinsequence for isolate NL/22/01 (A2) SEQ ID NO: 136 G-protein sequence forisolate NL/24/01 (A2) SEQ ID NO: 137 G-protein sequence for isolateNL/23/01 (A2) SEQ ID NO: 138 G-protein sequence for isolate NL/29/01(A2) SEQ ID NO: 139 G-protein sequence for isolate NL/3/02 (A2) SEQ IDNO: 140 G-protein sequence for isolate NL/1/99 (B1) SEQ ID NO: 141G-protein sequence for isolate NL/11/00 (B1) SEQ ID NO: 142 G-proteinsequence for isolate NL/12/00 (B1) SEQ ID NO: 143 G-protein sequence forisolate NL/5/01 (B1) SEQ ID NO: 144 G-protein sequence for isolateNL/9/01 (B1) SEQ ID NO: 145 G-protein sequence for isolate NL/21/01 (B1)SEQ ID NO: 146 G-protein sequence for isolate NL/1/94 (B2) SEQ ID NO:147 G-protein sequence for isolate NL/1/82 (B2) SEQ ID NO: 148 G-proteinsequence for isolate NL/1/96 (B2) SEQ ID NO: 149 G-protein sequence forisolate NL/6/97 (B2) SEQ ID NO: 150 G-protein sequence for isolateNL/9/00 (B2) SEQ ID NO: 151 G-protein sequence for isolate NL/3/01 (B2)SEQ ID NO: 152 G-protein sequence for isolate NL/4/01 (B2) SEQ ID NO:153 G-protein sequence for isolate NL/5/01 (B2) SEQ ID NO: 154 F-genecoding sequence for isolate NL/1/00 SEQ ID NO: 155 F-gene codingsequence for isolate UK/1/00 SEQ ID NO: 156 F-gene coding sequence forisolate NL/2/00 SEQ ID NO: 157 F-gene coding sequence for isolateNL/13/00 SEQ ID NO: 158 F-gene coding sequence for isolate NL/14/00 SEQID NO: 159 F-gene coding sequence for isolate FL/3/01 SEQ ID NO: 160F-gene coding sequence for isolate FL/4/01 SEQ ID NO: 161 F-gene codingsequence for isolate FL/8/01 SEQ ID NO: 162 F-gene coding sequence forisolate UK/1/01 SEQ ID NO: 163 F-gene coding sequence for isolateUK/7/01 SEQ ID NO: 164 F-gene coding sequence for isolate FL/10/01 SEQID NO: 165 F-gene coding sequence for isolate NL/6/01 SEQ ID NO: 166F-gene coding sequence for isolate NL/8/01 SEQ ID NO: 167 F-gene codingsequence for isolate NL/10/01 SEQ ID NO: 168 F-gene coding sequence forisolate NL/14/01 SEQ ID NO: 169 F-gene coding sequence for isolateNL/20/01 SEQ ID NO: 170 F-gene coding sequence for isolate NL/25/01 SEQID NO: 171 F-gene coding sequence for isolate NL/26/01 SEQ ID NO: 172F-gene coding sequence for isolate NL/28/01 SEQ ID NO: 173 F-gene codingsequence for isolate NL/30/01 SEQ ID NO: 174 F-gene coding sequence forisolate BR/2/01 SEQ ID NO: 175 F-gene coding sequence for isolateBR/3/01 SEQ ID NO: 176 F-gene coding sequence for isolate NL/2/02 SEQ IDNO: 177 F-gene coding sequence for isolate NL/4/02 SEQ ID NO: 178 F-genecoding sequence for isolate NL/5/02 SEQ ID NO: 179 F-gene codingsequence for isolate NL/6/02 SEQ ID NO: 180 F-gene coding sequence forisolate NL/7/02 SEQ ID NO: 181 F-gene coding sequence for isolateNL/9/02 SEQ ID NO: 182 F-gene coding sequence for isolate FL/1/02 SEQ IDNO: 183 F-gene coding sequence for isolate NL/1/81 SEQ ID NO: 184 F-genecoding sequence for isolate NL/1/93 SEQ ID NO: 185 F-gene codingsequence for isolate NL/2/93 SEQ ID NO: 186 F-gene coding sequence forisolate NL/4/93 SEQ ID NO: 187 F-gene coding sequence for isolateNL/1/95 SEQ ID NO: 188 F-gene coding sequence for isolate NL/2/96 SEQ IDNO: 189 F-gene coding sequence for isolate NL/3/96 SEQ ID NO: 190 F-genecoding sequence for isolate NL/1/98 SEQ ID NO: 191 F-gene codingsequence for isolate NL/17/00 SEQ ID NO: 192 F-gene coding sequence forisolate NL/22/01 SEQ ID NO: 193 F-gene coding sequence for isolateNL/29/01 SEQ ID NO: 194 F-gene coding sequence for isolate NL/23/01 SEQID NO: 195 F-gene coding sequence for isolate NL/17/01 SEQ ID NO: 196F-gene coding sequence for isolate NL/24/01 SEQ ID NO: 197 F-gene codingsequence for isolate NL/3/02 SEQ ID NO: 198 F-gene coding sequence forisolate NL/3/98 SEQ ID NO: 199 F-gene coding sequence for isolateNL/1/99 SEQ ID NO: 200 F-gene coding sequence for isolate NL/2/99 SEQ IDNO: 201 F-gene coding sequence for isolate NL/3/99 SEQ ID NO: 202 F-genecoding sequence for isolate NL/11/00 SEQ ID NO: 203 F-gene codingsequence for isolate NL/12/00 SEQ ID NO: 204 F-gene coding sequence forisolate NL/1/01 SEQ ID NO: 205 F-gene coding sequence for isolateNL/5/01 SEQ ID NO: 206 F-gene coding sequence for isolate NL/9/01 SEQ IDNO: 207 F-gene coding sequence for isolate NL/19/01 SEQ ID NO: 208F-gene coding sequence for isolate NL/21/01 SEQ ID NO: 209 F-gene codingsequence for isolate UK/11/01 SEQ ID NO: 210 F-gene coding sequence forisolate FL/1/01 SEQ ID NO: 211 F-gene coding sequence for isolateFL/2/01 SEQ ID NO: 212 F-gene coding sequence for isolate FL/5/01 SEQ IDNO: 213 F-gene coding sequence for isolate FL/7/01 SEQ ID NO: 214 F-genecoding sequence for isolate FL/9/01 SEQ ID NO: 215 F-gene codingsequence for isolate UK/10/01 SEQ ID NO: 216 F-gene coding sequence forisolate NL/1/02 SEQ ID NO: 217 F-gene coding sequence for isolateNL/1/94 SEQ ID NO: 218 F-gene coding sequence for isolate NL/1/96 SEQ IDNO: 219 F-gene coding sequence for isolate NL/6/97 SEQ ID NO: 220 F-genecoding sequence for isolate NL/7/00 SEQ ID NO: 221 F-gene codingsequence for isolate NL/9/00 SEQ ID NO: 222 F-gene coding sequence forisolate NL/19/00 SEQ ID NO: 223 F-gene coding sequence for isolateNL/28/00 SEQ ID NO: 224 F-gene coding sequence for isolate NL/3/01 SEQID NO: 225 F-gene coding sequence for isolate NL/4/01 SEQ ID NO: 226F-gene coding sequence for isolate NL/11/01 SEQ ID NO: 227 F-gene codingsequence for isolate NL/15/01 SEQ ID NO: 228 F-gene coding sequence forisolate NL/18/01 SEQ ID NO: 229 F-gene coding sequence for isolateFL/6/01 SEQ ID NO: 230 F-gene coding sequence for isolate UK/5/01 SEQ IDNO: 231 F-gene coding sequence for isolate UK/8/01 SEQ ID NO: 232 F-genecoding sequence for isolate NL/12/02 SEQ ID NO: 233 F-gene codingsequence for isolate HK/1/02 SEQ ID NO: 234 F-protein sequence forisolate NL/1/00 SEQ ID NO: 235 F-protein sequence for isolate UK/1/00SEQ ID NO: 236 F-protein sequence for isolate NL/2/00 SEQ ID NO: 237F-protein sequence for isolate NL/13/00 SEQ ID NO: 238 F-proteinsequence for isolate NL/14/00 SEQ ID NO: 239 F-protein sequence forisolate FL/3/01 SEQ ID NO: 240 F-protein sequence for isolate FL/4/01SEQ ID NO: 241 F-protein sequence for isolate FL/8/01 SEQ ID NO: 242F-protein sequence for isolate UK/1/01 SEQ ID NO: 243 F-protein sequencefor isolate UK/7/01 SEQ ID NO: 244 F-protein sequence for isolateFL/10/01 SEQ ID NO: 245 F-protein sequence for isolate NL/6/01 SEQ IDNO: 246 F-protein sequence for isolate NL/8/01 SEQ ID NO: 247 F-proteinsequence for isolate NL/10/01 SEQ ID NO: 248 F-protein sequence forisolate NL/14/01 SEQ ID NO: 249 F-protein sequence for isolate NL/20/01SEQ ID NO: 250 F-protein sequence for isolate NL/25/01 SEQ ID NO: 251F-protein sequence for isolate NL/26/01 SEQ ID NO: 252 F-proteinsequence for isolate NL/28/01 SEQ ID NO: 253 F-protein sequence forisolate NL/30/01 SEQ ID NO: 254 F-protein sequence for isolate BR/2/01SEQ ID NO: 255 F-protein sequence for isolate BR/3/01 SEQ ID NO: 256F-protein sequence for isolate NL/2/02 SEQ ID NO: 257 F-protein sequencefor isolate NL/4/02 SEQ ID NO: 258 F-protein sequence for isolateNL/5/02 SEQ ID NO: 259 F-protein sequence for isolate NL/6/02 SEQ ID NO:260 F-protein sequence for isolate NL/7/02 SEQ ID NO: 261 F-proteinsequence for isolate NL/9/02 SEQ ID NO: 262 F-protein sequence forisolate FL/1/02 SEQ ID NO: 263 F-protein sequence for isolate NL/1/81SEQ ID NO: 264 F-protein sequence for isolate NL/1/93 SEQ ID NO: 265F-protein sequence for isolate NL/2/93 SEQ ID NO: 266 F-protein sequencefor isolate NL/4/93 SEQ ID NO: 267 F-protein sequence for isolateNL/1/95 SEQ ID NO: 268 F-protein sequence for isolate NL/2/96 SEQ ID NO:269 F-protein sequence for isolate NL/3/96 SEQ ID NO: 270 F-proteinsequence for isolate NL/1/98 SEQ ID NO: 271 F-protein sequence forisolate NL/17/00 SEQ ID NO: 272 F-protein sequence for isolate NL/22/01SEQ ID NO: 273 F-protein sequence for isolate NL/29/01 SEQ ID NO: 274F-protein sequence for isolate NL/23/01 SEQ ID NO: 275 F-proteinsequence for isolate NL/17/01 SEQ ID NO: 276 F-protein sequence forisolate NL/24/01 SEQ ID NO: 277 F-protein sequence for isolate NL/3/02SEQ ID NO: 278 F-protein sequence for isolate NL/3/98 SEQ ID NO: 279F-protein sequence for isolate NL/1/99 SEQ ID NO: 280 F-protein sequencefor isolate NL/2/99 SEQ ID NO: 281 F-protein sequence for isolateNL/3/99 SEQ ID NO: 282 F-protein sequence for isolate NL/11/00 SEQ IDNO: 283 F-protein sequence for isolate NL/12/00 SEQ ID NO: 284 F-proteinsequence for isolate NL/1/01 SEQ ID NO: 285 F-protein sequence forisolate NL/5/01 SEQ ID NO: 286 F-protein sequence for isolate NL/9/01SEQ ID NO: 287 F-protein sequence for isolate NL/19/01 SEQ ID NO: 288F-protein sequence for isolate NL/21/01 SEQ ID NO: 289 F-proteinsequence for isolate UK/11/01 SEQ ID NO: 290 F-protein sequence forisolate FL/1/01 SEQ ID NO: 291 F-protein sequence for isolate FL/2/01SEQ ID NO: 292 F-protein sequence for isolate FL/5/01 SEQ ID NO: 293F-protein sequence for isolate FL/7/01 SEQ ID NO: 294 F-protein sequencefor isolate FL/9/01 SEQ ID NO: 295 F-protein sequence for isolateUK/10/01 SEQ ID NO: 296 F-protein sequence for isolate NL/1/02 SEQ IDNO: 297 F-protein sequence for isolate NL/1/94 SEQ ID NO: 298 F-proteinsequence for isolate NL/1/96 SEQ ID NO: 299 F-protein sequence forisolate NL/6/97 SEQ ID NO: 300 F-protein sequence for isolate NL/7/00SEQ ID NO: 301 F-protein sequence for isolate NL/9/00 SEQ ID NO: 302F-protein sequence for isolate NL/19/00 SEQ ID NO: 303 F-proteinsequence for isolate NL/28/00 SEQ ID NO: 304 F-protein sequence forisolate NL/3/01 SEQ ID NO: 305 F-protein sequence for isolate NL/4/01SEQ ID NO: 306 F-protein sequence for isolate NL/11/01 SEQ ID NO: 307F-protein sequence for isolate NL/15/01 SEQ ID NO: 308 F-proteinsequence for isolate NL/18/01 SEQ ID NO: 309 F-protein sequence forisolate FL/6/01 SEQ ID NO: 310 F-protein sequence for isolate UK/5/01SEQ ID NO: 311 F-protein sequence for isolate UK/8/01 SEQ ID NO: 312F-protein sequence for isolate NL/12/02 SEQ ID NO: 313 F-proteinsequence for isolate HK/1/02 SEQ ID NO: 314 F protein sequence for HMPVisolate NL/1/00 SEQ ID NO: 315 F protein sequence for HMPV isolateNL/17/00 SEQ ID NO: 316 F protein sequence for HMPV isolate NL/1/99 SEQID NO: 317 F protein sequence for HMPV isolate NL/1/94 SEQ ID NO: 318F-gene sequence for HMPV isolate NL/1/00 SEQ ID NO: 319 F-gene sequencefor HMPV isolate NL/17/00 SEQ ID NO: 320 F-gene sequence for HMPVisolate NL/1/99 SEQ ID NO: 321 F-gene sequence for HMPV isolate NL/1/94SEQ ID NO: 322 G protein sequence for HMPV isolate NL/1/00 SEQ ID NO:323 G protein sequence for HMPV isolate NL/17/00 SEQ ID NO: 324 Gprotein sequence for HMPV isolate NL/1/99 SEQ ID NO: 325 G proteinsequence for HMPV isolate NL/1/94 SEQ ID NO: 326 G-gene sequence forHMPV isolate NL/1/00 SEQ ID NO: 327 G-gene sequence for HMPV isolateNL/17/00 SEQ ID NO: 328 G-gene sequence for HMPV isolate NL/1/99 SEQ IDNO: 329 G-gene sequence for HMPV isolate NL/1/94 SEQ ID NO: 330 Lprotein sequence for HMPV isolate NL/1/00 SEQ ID NO: 331 L proteinsequence for HMPV isolate NL/17/00 SEQ ID NO: 332 L protein sequence forHMPV isolate NL/1/99 SEQ ID NO: 333 L protein sequence for HMPV isolateNL/1/94 SEQ ID NO: 334 L-gene sequence for HMPV isolate NL/1/00 SEQ IDNO: 335 L-gene sequence for HMPV isolate NL/17/00 SEQ ID NO: 336 L-genesequence for HMPV isolate NL/1/99 SEQ ID NO: 337 L-gene sequence forHMPV isolate NL/1/94 SEQ ID NO: 338 M2-1 protein sequence for HMPVisolate NL/1/00 SEQ ID NO: 339 M2-1 protein sequence for HMPV isolateNL/17/00 SEQ ID NO: 340 M2-1 protein sequence for HMPV isolate NL/1/99SEQ ID NO: 341 M2-1 protein sequence for HMPV isolate NL/1/94 SEQ ID NO:342 M2-1 gene sequence for HMPV isolate NL/1/00 SEQ ID NO: 343 M2-1 genesequence for HMPV isolate NL/17/00 SEQ ID NO: 344 M2-1 gene sequence forHMPV isolate NL/1/99 SEQ ID NO: 345 M2-1 gene sequence for HMPV isolateNL/1/94 SEQ ID NO: 346 M2-2 protein sequence for HMPV isolate NL/1/00SEQ ID NO: 347 M2-2 protein sequence for HMPV isolate NL/17/00 SEQ IDNO: 348 M2-2 protein sequence for HMPV isolate NL/1/99 SEQ ID NO: 349M2-2 protein sequence for HMPV isolate NL/1/94 SEQ ID NO: 350 M2-2 genesequence for HMPV isolate NL/1/00 SEQ ID NO: 351 M2-2 gene sequence forHMPV isolate NL/17/00 SEQ ID NO: 352 M2-2 gene sequence for HMPV isolateNL/1/99 SEQ ID NO: 353 M2-2 gene sequence for HMPV isolate NL/1/94 SEQID NO: 354 M2 gene sequence for HMPV isolate NL/1/00 SEQ ID NO: 355 M2gene sequence for HMPV isolate NL/17/00 SEQ ID NO: 356 M2 gene sequencefor HMPV isolate NL/1/99 SEQ ID NO: 357 M2 gene sequence for HMPVisolate NL/1/94 SEQ ID NO: 358 M protein sequence for HMPV isolateNL/1/00 SEQ ID NO: 359 M protein sequence for HMPV isolate NL/17/00 SEQID NO: 360 M protein sequence for HMPV isolate NL/1/99 SEQ ID NO: 361 Mprotein sequence for HMPV isolate NL/1/94 SEQ ID NO: 362 M gene sequencefor HMPV isolate NL/1/00 SEQ ID NO: 363 M gene sequence for HMPV isolateNL/17/00 SEQ ID NO: 364 M gene sequence for HMPV isolate NL/1/99 SEQ IDNO: 365 M gene sequence for HMPV isolate NL/1/94 SEQ ID NO: 366 Nprotein sequence for HMPV isolate NL/1/00 SEQ ID NO: 367 N proteinsequence for HMPV isolate NL/17/00 SEQ ID NO: 368 N protein sequence forHMPV isolate NL/1/99 SEQ ID NO: 369 N protein sequence for HMPV isolateNL/1/94 SEQ ID NO: 370 N gene sequence for HMPV isolate NL/1/00 SEQ IDNO: 371 N gene sequence for HMPV isolate NL/17/00 SEQ ID NO: 372 N genesequence for HMPV isolate NL/1/99 SEQ ID NO: 373 N gene sequence forHMPV isolate NL/1/94 SEQ ID NO: 374 P protein sequence for HMPV isolateNL/1/00 SEQ ID NO: 375 P protein sequence for HMPV isolate NL/17/00 SEQID NO: 376 P protein sequence for HMPV isolate NL/1/99 SEQ ID NO: 377 Pprotein sequence for HMPV isolate NL/1/94 SEQ ID NO: 378 P gene sequencefor HMPV isolate NL/1/00 SEQ ID NO: 379 P gene sequence for HMPV isolateNL/17/00 SEQ ID NO: 380 P gene sequence for HMPV isolate NL/1/99 SEQ IDNO: 381 P gene sequence for HMPV isolate NL/1/94 SEQ ID NO: 382 SHprotein sequence for HMPV isolate NL/1/00 SEQ ID NO: 383 SH proteinsequence for HMPV isolate NL/17/00 SEQ ID NO: 384 SH protein sequencefor HMPV isolate NL/1/99 SEQ ID NO: 385 SH protein sequence for HMPVisolate NL/1/94 SEQ ID NO: 386 SH gene sequence for HMPV isolate NL/1/00SEQ ID NO: 387 SH gene sequence for HMPV isolate NL/17/00 SEQ ID NO: 388SH gene sequence for HMPV isolate NL/1/99 SEQ ID NO: 389 SH genesequence for HMPV isolate NL/1/94

What is claimed is:
 1. A method for determining the presence ofmetapneumovirus (MPV) in a mammalian subject, the method comprising:contacting a sample from the subject comprising the targetpolynucleotide with a DNA probe of at least 10 nucleotides thathybridizes specifically to target polynucleotide; wherein the targetpolynucleotide comprises a sequence encoding a polypeptide that is atleast 90% identical to SEQ ID NO:366 or the complement of the sequence;hybridizing the DNA probe and the target polynucleotide to form acomplex; and detecting the complex.
 2. The method according to claim 1,wherein the DNA probe does not specifically hybridize to apolynucleotide from avian pneumovirus (APV).
 3. The method according toclaim 1, wherein the DNA probe does specifically hybridize to apolynucleotide from avian pneumovirus (APV).
 4. The method according toclaim 1, wherein the DNA probe comprises at least 25 nucleotides.
 5. Themethod according to claim 1, wherein the DNA probe comprises at least 40nucleotides.
 6. The method according to claim 1, wherein detecting thecomplex comprises performing PCR with the DNA probe as a primer.
 7. Themethod according to claim 1, wherein the mammalian subject is a human.8. The method according to claim 1, wherein the DNA probe comprises adetectable marker.
 9. The method according to claim 1, wherein the DNAprobe is attached to a solid support.
 10. The method according to claim1, wherein the target polynucleotide comprises a nucleic acid encodingSEQ ID NO:366.
 11. The method according to claim 1, wherein the targetpolynucleotide comprises the complement of a nucleic acid encoding SEQID NO:366.
 12. A kit for determining the presence of metapneumovirus(MPV) in a mammalian subject, the kit comprising: a DNA probe of atleast 10 nucleotides that specifically hybridizes to a targetpolynucleotide, wherein the target polynucleotide comprises a sequenceencoding a polypeptide that is at least 90% identical to SEQ ID NO:366or the complement of the sequence, and wherein the DNA probe does notspecifically hybridize to a polynucleotide from avian pneumovirus (APV).13. The kit of claim 12, wherein the DNA probe comprises at least 25nucleotides.
 14. The kit of claim 12, wherein the DNA probe comprises atleast 40 nucleotides.
 15. The kit of claim 12, wherein the DNA probefurther comprises detectable marker.
 16. The kit of claim 12, whereinthe target polynucleotide comprises a nucleic acid encoding SEQ IDNO:366.
 17. The method according to claim 12, wherein the targetpolynucleotide comprises the complement of a nucleic acid encoding SEQID NO:366.